Plant Response as a Means of Physiological Investigation

In this experiment, then, the root having been cut oft", there is still a considerable propulsion of water upwards through the stem. It is thus clear that root-pressure is not the essential factor in the ascent of sap. As the leaves had also been cut off, and the cut end of the stem covered by water sealed with a film of oil, evaporation from the leaves, and the osmotic action thereby produced, are also seen to be excluded. These, like root-pressure, therefore, cannot constitute the essential factors in the process of suction. But since, on the contrary, any small length of the stem is competent to show this water-movement, the required activity must reside in the tissue of the stem.

A balance was finally obtained, by the pressure of a water-column of 105 cm. This equilibrium is not to be regarded as merely the result of equality between the upward pressure, exerted by suction from below, and the downward pressure of the superincumbent water-column. There are reasons for thinking that there may also be an important additional factor, of opposed rhythmic activities, balancing each other. For when a cut branch is placed in water, the lower end becomes over-turgid, and this, as we know, is a condition for the initiation of rhythmic activity. The upper end of the branch not being so turgid, activity will be greater below than above. We therefore obtain a movement of water from the more to the less active. But if the leafy end of the branch be immersed in water, and the cut end held in the air, it is known that the direction of the flow becomes reversed. This is evidently due to the fact that it is now the upper end which is over-turgid, and therefore relatively the more active. Similarly, in the case of the balance described, the activity of the lower end, which determines the flow upwards, was opposed and balanced by the increased activity of the upper half, induced by increased hydrostatic pressure.1 The experiment which I am about to describe, besides demonstrating the effects of cold and warmth, also lends strong support to the view that the direction of the resultant movement of sap is determined by the relative activities of the two ends of the stem.

When the balance had been obtained, as already described, with a pressure of water of 105 cm., the record on the drum became horizontal, as has been explained. Cold water was now applied to the specimen at its lower end. As sudden cooling constitutes a stimulus, while its continued action produces depression, we should expect a transient augmentation of the activity of the lower end of the specimen, followed by its diminution and arrest. The record should therefore show a preliminary movement of water upwards, followed by the reversal of the current, which should now, as the permanent effect of cold applied below, be from the

1 Rhythmic activity is, in general, increased by an increase in the hydrostatic pressure. But there is a limit to this. Excessive pressure, above a certain critical point, is found to depress rhythmic activity (p. 350). more active upper, to the less active lower end, or downwards. The record will be seen to verify this anticipation in every particular (fig. 161). Cold water was applied below — represented by x in the record — which before application was horizontal. After this we observe^ a movement of water upwards, at a rate of four cubic mm. per minute. The upward flow continues for about seven minutes, by which time the

Fig. 161. Record obtained by Method of Hydrostatic Balance of Successive Applications of Cold and Warm Water After obtaining static balance, with horizontal record not shown in the figure, cold water was applied below at moment x . The transitory excitation thus occasioned causes movement of water upwards, followed by depression and reversal of flow downwards. Warm water next applied at f causes second reversal and flow of water upwards. activity of the lower end of the specimen has become so depressed as to cause reversal of the flow, which is now from above downwards at an average rate of about six cubic mm. per minute. This record was taken continuously for some time, when hot water was quickly substituted for cold, in the plant-vessel. A cross marks this point in the record.

its sluggishness was now obviated, but that the lower end of the specimen was actually rendered the more excitable of the two, and we observe a second reversal of the direction of current, which now flows upwards. (3) Method of Hydraulic Balance.— This Hydraulic Method of Balance is much easier to carry out than the arrest of movement by hydrostatic pressure. It does not, moreover, in any way interfere with the normal movement of water through the plant. For the balance is obtained and the index rendered stationary, by the simple device already explained, of allowing a subsidiary flow of water to enter the plant-vessel, at a rate exactly sufficient to compensate for the loss by ascent of sap.

(a) Action of cold. — This experiment was performed on a leafy branch of Croton. The balanced horizontal record was first taken at 220 C, after which ice-cold water was passed into the vessel. A record was made of the immediate or preliminary excitatory effect of this cold water on the rate of suction, and continued during the return of the water to the temperature of the room. In this record, then, we shall find, besides the immediate, the continued effect of cold, and subsequently the effect of gradual restoration to an ordinary temperature. In the present record (fig. 162) we see the transient and permanent effects of cold exhibited as before. According to the method of Hydraulic Balance, as has been explained, an ascending line in the record means a positive variation, or increase of the rate of suction over the normal. A descending line, on the contrary, denotes a negative variation, or diminution of the rate of suction below the normal. And a horizontal line shows return to the original rate. In the first, or upper, of the two curves in fig. 162, the immediate effect of cold is seen in a very marked positive variation. After the lapse of about five minutes, the effect of continued cold is seen in the depression, which shows itself by the reversal of the curve. This depression continues, till the temperature of the vessel has returned to that of the room, which takes place in the course of about forty minutes.

The normal rate of suction is now re-established, as seen in the fact that the record becomes horizontal. (J?) Effect of warm water. — I next tried the effect on the same specimen of water at a higher temperature, falling by degrees to the temperature of the room. We should expect in such a case, to obtain the excitatory effect of rise of temperature, with subsequent approach to the normal, without any reversal, indicative of the transition from exaltation to depression, such as was observed in the case of application of cold. From the second and lower of the two curves

FlG. 162. Record, obtained by Method of Hydraulic Balance, of Successive Effects of Cold and Warm Water shown in fig. 162, it will be seen that this is the case. The curve at first rises abruptly, and it continues to rise, though with decreased speed, till, when the temperature of the vessel is once more normal, it becomes horizontal, indicating the resumption of the original rate of suction. We have thus seen, by three different methods of inquiry — those namely of the Unbalanced Shoshungraph, the Hydrostatic Balance, and the Hydraulic Balance— that the records, though of different forms, exhibit effects of which the interpretations are identical. For delicate experiments, the

Hydraulic Method of Balance will be found most sensitive : for ordinary purposes, however, the unbalanced Shoshungraph is simple and efficient ; and in the investigations which follow I shall use this method only. Explanation of suction, when the root is killed by boiling water. — I shall now take up the apparently anomalous case in which, when the root has been killed, by pouring boiling water over it, the suction of the plant is nevertheless maintained. In such an experiment, the normal record was first taken, and on allowing boiling water to enter the plantchamber there was a steep rise in the record, showing the excitatory action due to the application of hot water. The boiling water was now passed in continuously for several minutes, so as to ensure the killing of that portion of the plant which was immersed in the vessel. On allowing the water in the vessel to return to the temperature of the room, it was found that suction continued, at an even greater than the original normal rate.

This result would at first appear to show that protoplasmic activity had nothing to do with the ascent of sap. And the objection would have been fatal, if the rhythmic activity which produces suction had been confined to the roots alone. But such activity is present to a greater or less extent throughout every zone of the plant, and it is by the combined action of all these that the ascensional movement is maintained (p. 376). Thus, when hot water is poured on the root, its first effect is a sudden increase of the activity of that organ, by which warm water is carried to the higher zones, there as a stimulating agency to increase this rhythmic activity. It must be remembered that on reaching the stem above the vessel, the hot water itself is considerably cooled. Hence the only portion of the plant which is killed is that which is actually immersed in the boiling water, or in immediate contiguity with it. The unkilled portions above continue their suctional activity unabated.

I have said that the suction, on the return of the water to its old temperature, continued to take place at a greater than the original rate. This was due to the fact that, instead of the extremely attenuated channels of the root-hairs, through which suction normally takes place, there was now substituted the whole mass of the root, acting virtually as a wet rag, tied round the base of the living stem ; and indeed it was found that, whereas the stem outside the vessel was turgid, the portion within was limp and soft. The mass of water which it was thus possible to suck up directly, by means of the broad-sectioned stem, was evidently much greater than could have been the case through the intervention of the resistant organically conducting channels of the rootlets.

We must not forget the obvious fact that a plant is a colony of more or less independent living cells, each of which maintains its physiological activity as an individual. The death of one group does not necessarily, therefore, arrest the physiological activity of its neighbours. The plant is finally killed only when every one of its cellular elements has undergone death. Further proof that suction is an excitatory response.— We have seen in the case of rhythmic Desmodium, when it is kept for a long time under unfavourable circumstances, that its activity comes to a stop owing to the run-down of stored-up energy. We also saw how the application of thermal stimulus would re-initiate this activity. Again, if we keep a cut branch of any plant in water, after a few days its suctional activity, as is well known, disappears. This abolition of suction is attributed to the blocking of the cut end by mucilage and bacterial growths, and the making of a fresh section is found to renew the activity.

But, though the blocking of the cut end of the stem by outgrowths does, no doubt, obstruct the passage of water, yet the total abolition of suction may not be due to this cause alone. It may be induced, in part at least, by depression of the rhythmic activity of the tissue, owing to the rundown of its latent energy. The making of a fresh section does not decide this question, for, in doing this, we apply the strong mechanical stimulus of a cut. I therefore devised an experiment which appears to show that this run-down of energy does in such a case constitute a factor in the abolition of suction. Without in any way disturbing the mucilaginous end of the stem, which had ceased to exhibit its suctional activity, I supplied it with water somewhat above the ordinary temperature. This thermal stimulation at once initiated renewed suctional activity with great vigour, just as its rhythmic mechanical activity was renewed by Desmodium on the application of similar stimulus.

Osmotic versus excitatory action.— Though, under the co-operation of a favourable disposition of osmotic substances, the suctional activity of the tissue may be increased, yet I have shown that suction is normally maintained even without the co-operation of this factor (p. 376). I shall now proceed to show that this suction may increase even in opposition to osmotic action. And such a demonstration will further prove the excitatory physiological nature of the processes which bring about the ascent of sap. Among various solutions of salt, some are physiologically neutral in their effects ; l of these, potassium nitrate may be taken as an example. Others, again, like strong solutions of sodium chloride, act as excitatory agents. The application of this last reagent is known to initiate rhythmic excitation in animal tissues. Similar effects have been shown to be brought about by this reagent, in the case of Biophytum and Desmodium.

Thus, in a strong solution of potassium nitrate, we have a reagent whose physiological action is more or less neutral while its osmotic action is pronounced, and in a strong solution of common salt we have an agent which is both excitatory and osmotic at the same time. If, then, we apply KNO3 solution to the base of a cut stem, placed in the Shoshungraph, water will be osmotically withdrawn from the plant, in opposition to normal suction, and the

1 It should, however, be remembered that solutions, even of inactive salts, above 'a certain strength, will induce physiological depression, and thus bring about diminution of transpiration. normal suctional rate will be somewhat reduced. This is seen in the accompanying record (fig. 163), which I obtained with a cut branch of Croton. The normal rate of suction was in this case twenty-six cubic mm. per minute. After the application of potassium nitrate solution this was found to be reduced to seventeen cubic mm. per minute. But if, instead of this, we apply strong solution of sodium chloride, two antagonistic effects will be produced. One, due to

The first record shows the normal and the second the depressed rate of suction caused by the reagent. The first record shows the normal and the second the exalted rate of suction caused by the reagent. osmotic action, will oppose suction ; and the other, due to the excitatory nature of the reagent, will accelerate suction. The resultant effect will, then, be modified by the excitability of the experimental plant itself. In some cases we should expect to find the excitatory reaction predominant, and in others the osmotic. In repeating the experiment, on different specimens of Croton, I have found these theoretical inferences to be fully verified. As the more interesting of the two cases, I give a record (fig. 164) in

which the excitatory effect is shown by the very great increase in the rate of suction induced after the application of the reagent. We have here a very great enhancement of the ascensional movement of water in spite of the osmotic attraction of the solution, which alone would have retarded the normal rate. The action of poisonous reagents. — In studying the effect of poison on the rhythmic activity of Desmodium, we found that this was modified by the tonic condition of the plant. Thus a vigorous specimen was shown to be much less affected by poison than one which was weakly. Certain poisons, again, act more quickly than others in inducing the death of the plant. We have also seen, in that experiment in which the root was killed with hot water, that upper and unkilted portions of a specimen will continue to exhibit suctional activity when lower parts are killed ; and

also that, in general, the killed area offers no barrier to the That a poison can easily pass through killed tissues, owing to the suctional activity of cells higher up, we have seen in our experiments on Desmodium, when the cut end of the petiole was placed in copper sulphate solution (p. 326). It is fortunate that in this case, during the ascent of poison, we have areas, the activity of which is indicated visibly by the rhythmic motile indications of the pulvini of the inserted lateral, leaflets. That copper sulphate solution arrests rhythmic activity and induces death, is seen by the rapid stoppage of pulsation, when we apply it directly on the pulvini of the pulsating leaflets. When it is applied, however, at the cut end of the petiole, the arrest of pulsation takes place much later, this delay being due to the time taken for the poison to ascend through the intervening distance. This shows clearly that successive zones are killed one after another, and that the death of a point below does not stop the suction above. From this experiment it is evident that the application of poison at the root, or the cut end of a stem, does not in general arrest

suction, until the whole plant is killed. And from the Shoshungraphic records we find that the final arrest occurs after an appropriately long period. In this connection, I shall describe some very interesting results of rapid arrest of suction, which I have often obtained by the action of poison. I was already familiar with a fact which I had come across while studying the effects of various chemical reagents on the longitudinal response of radial organs namely, that death was attended in such cases either by an abnormal contraction or by an abnormal relaxation. These two effects were liable, again, to be modified by the

The first part of the record shows the normal rate 01 suction. The asterisk denotes the time of application of the poisonous reagent. tonic condition of the tissue. In now studying the effect of solution of copper sulphate on the suctional activity of Crolony I noticed certain peculiarities in the record, which appeared to be related to the results just described. These peculiarities, it should be mentioned, were specially noticeable in those specimens which were experimented on during the month of April, that is to say, at the end of the Indian spring.

In a particular experiment the normal suctional rate had been fifteen cubic mm. per minute. On the application of copper sulphate, the suctional movement was quickly arrested, and this was followed almost immediately by a slight movement in the negative direction, showing that, by some spasmodic contraction, water was being expelled from the tissue. This phase was succeeded by an almost complete arrest of suction, there being now only the feeblest ascensional movement (fig. 165). Within a short period after this, on washing off the poisonous reagent, it was found that the arrest had been temporary only, suction being renewed at the rate of eleven, instead of the normal fifteen, cubic mm. per minute.

I applied the poison once more, and allowed it to act for thirty-six hours. The arrest was then found to be permanent — that is to say, the substitution of fresh water induced no revival of response, the plant being killed throughout. Strasburger, as we have seen, in his experiments on the effect of poisonous reagents on plants, found that the reagent is carried to the top of the tallest tree ; from this fact it was inferred that since all the cells in the path of the poisonous solution must necessarily be killed by its action, therefore the activity of living cells was not the essential factor in the ascent of sap. But I have proved that the ascent of sap is brought about, not by any localised group of cells in a particular region, but by cells which extend throughout the length of the plant. Even after some of these have died, therefore, by the access of poison, those above are still active, and will continue to exhibit suction till they in their turn are finally killed. It will thus be evident that the movement of ascent cannot be completely abolished till the poison has effectively reached the very top. As all the living cells are actively concerned in the work of suction, this conveyance of poison to the top of the plant is what was to be expected. Only after such conveyance, indeed, could permanent arrest possibly take place, and, in fact, Strasburger himself mentions that the movement of water did come to a stop when the poison reached the top of the tree.

Rhythmic activity being as a rule -exalted by rise of temperature, suctional response, as one of its effects, also undergoes an increase. Suction being an expression of excitatory response, the direction of the resultant movement of sap is determined by the relative excitabilities of the two ends of a tissue. Under certain circumstances, the normal direction of movement of sap may be reversed. The application of cold produces a transient excitation, and thus causes a preliminary enhancement of suction. Prolonged application of cold, causing a depression of excitability, brings about arrest of suction.

As suction is produced by the rhythmic activity of the tissue of the entire plant, local death, as by scalding or application of poison, does not cause its arrest, until the whole plant is killed. When the sum total of the latent energy of the tissue — that is to say, its tonic condition — is below par, its rhythmic suctional activity comes to a stop ; fresh application of stimulus, however, renews this activity. Osmotic substances, as regards their stimulatory action, may be either neutral or excitatory. If such a solution be applied at the root, there will in the former case be a diminution, and in the latter, if the excitatory action be relatively great, an increase of suction.

The application of poison abolishes local excitability and power of suction. In some cases this arrest of suction may occur quickly. But the total abolition of suction by poison only takes place, for reasons already explained, on the death of the plant as a whole. The mechanics of the ascent of sap : (a) Uni-directioned flow-(^) Initiation of multiple rhythmic excitations — Connection between conduction of excitation and conduction of sap — Rapidity of ascent of sap accounted for by stimulatory action — Positive and negative pressures clue to one cause — ( I ) Positive pressure — (2) Negative pressure — (3) Irregular variations of pressure — Direct conduction and conduction by relays— Excretion of water — Excretion of nectar — Translocation of organic food-substances — Mechanical response to suctional activity -Effect of warmth — Effect of cold — Explanation of the drooping of leaves during frost — Explanation of response and recovery — Antagonistic actions of internal energy and external stimulus.

THERE are various phenomena connected with the transport of water in plants, which are at present considered as entirely distinct. Thus, for example, when a plant is cut above the root, the exuding water exerts a considerable positive pressure on a manometer, this being known as root or exudation pressure. But when manometers are inserted in lateral holes bored in the trunk of a tree, a negative pressure is observed. These facts have led to the inference that there exist, in a transpiring plant, two independent forces, one of suction, and the other of pressure. The negative pressure, again, which is, generally speaking, maximal at the top of a tree, falls to a minimal value near the root. But this fall is characterised by very irregular fluctuations, the negative pressure, in a zone below, being sometimes greater than that at a given distance above.

I shall now proceed to show, however, that these very various results are not actually due, as supposed, to the operation of distinct forces, but are, on the contrary, so many different effects, under different conditions, of the rhythmic cellular activity of the plant-tissue. I shall show that this activity is sufficient to account not only for the phenomena of the excretion of solution from pores and nectaries, but also for other phenomena, whose connection with it has been little suspected.

The mechanics of the ascent of sap. — We have seen that throughout the plant there are active rhythmic tissues, which by their excitatory activity bring about the movement of water. We have next to inquire, therefore, as to how this excitatory action is initiated, and further in what manner so many activities in different zones of the stem are correlated, so as to give rise to a uni-directional flow, generally upwards ; for the rhythmic activity which may cause .any given group of cells to act as a pump, would not alone be sufficient to account for the regulated, one-directioned flow of sap, since, while one such group propels water in one direction, there is no obvious reason why another should not propel it in the opposite.