The Physiology of the Ascent of Sap

ments, to the suctional activity of the living tissues beyond and above the poisoned region. That liquid, even poison- ous liquid, can travel through dead tissue is proved by the experiment v^'ith Desmodimn, described on p. 22, as also by others. The killed tissue of the poisoned stem becomes passive ; but so long as the parts above remain alive, it continues to exhibit suction, which is completely Fig. 26b. Curves of Suction of a in Water, and b in Poisonous Solution. Ordinate represents Quantity of Water sucked, and the Abscissa, the Time

Note that the slope of the curve a remains unchanged for days, indicating continuous suction (the portion of the curve after 2 p.m. has been omitted), while the curve b shows con- tinuous diminution and final abolition of suction. abolished only when the stem is killed throughout its entire length. It would take a long time to kill a large tree by applying poison to the root or to the cut surface of the trunk. Incidental reference may be made to Strasburger's scalding experiments. The account of the following experiment will be of interest. The root of a plant was killed by boiling water. On return to the normal tem-

perature, the suction of water, instead of being arrested, was found to be enhanced even above the normal. This result does not in any way invalidate the physiological theory ; for it was only the portion of the plant killed by boiling water which lost its activity, whereas that of the unkilled portion above remained unaffected. The greater rate of suction after killing of the root is due to the fact that instead of the extremely attenuated channels of the root- hairs through which water was previously absorbed, there was now substituted the broad-sectioned stem, the dead mass of the roots acting as a piece of moist cloth for supply- ing water to the living tissue.

The experimental evidence condensed in the following table proves that, under certain physiological changes, the ascent of sap undergoes variations which are identical with those of other forms of rhythmic activity, and justifies the conclusion that it is, like them, a rhythmic pheno- menon carried on by essentially the same mechanism — that of living pulsating cells. Showing the Effect of Physiological Changes on all Forms of Rhythmic Activity Diminished internal hydrostatic pressure induces a depression of the ascent of sap.

In sub-tonic specimens stimulus causes an enhancement of the rate of ascent ; in normal specimens it induces a retardation or arrest of ascent. Rise of temperature enhances the rate of ascent, while fall of temperature depresses it. The ascent is arrested at a temperature below the critical point ; an identical tissue can thus be made a con- ductor or non-conductor of the sap by raising or lowering the temperature above or below the critical point.

In the tropics the critical temperature-minimum is higher than in colder climates. The following relates to the critical point in several tropical plants : — The critical point of the fully grown pulvinule of Desmodium is about 14° C, which is the same as the critical point of ascent of sap in the cut stem of many plants : in growing organs, growth-activity is arrested even at the relatively high temperature of 22° C. : absorption and the ascent of sap in rooted plants are also arrested at about 22° C.

The effect of small doses of anaesthetics is to enhance the rhythmic activity of Desmodium leaflet, of growth, and of the ascent of sap. Large doses induce an arrest. Poisons arrest the ascent of sap in specimens with roots and also in cut shoots. These experimental results prove conclusively that it is the pulsatory activity of living cells which maintains the ascent of sap in the plant. Physical evaporation and physiological excretion — Isolation of absorbing, conducting, and excreting organs — The Bubbling Method for measure- ment of transpiration — Comparison of transpiring activity of different species of plants — Ratio of transpiration from upper and lower surfaces of leaves — Determination of transpiration from a single stoma — Transpiration in the absence of evaporation — The role of evaporation — Physiological continuity in stem and leaf — Crucial tests of physio- logical activity underlying transpiration — Effect of variation of temperature — Effects of sub-minimal and maximal stimulus — Summary.

The study of the ascent of the sap in the stem has shown that it is effected by an independent activity of its own : that it takes place in the absence of a root to propel the sap or of leaves to suck it (p. 36) . The direction of propa- gation is determined, as we have seen (p. 34), by the turgor- gradient, from the more turgid to the less turgid region of the plant. M^e have found that the activity of the root is not specifically different from that of the shoot, for the modifi- cation of the ascent of sap under physiological variation is essentially similar in cut stems and in intact plants with roots. We have arrived at the conclusion that the various manifestations of the ascent are brought about by the co-operative activity of living cells throughout the length of the plant, the absorbing root and the conducting stem. It now remains to study in detail the excretion of water at the upper end of the plant by the transpiring leaves. This is important, inasmuch as the state of turgor, internal pressure, exudation, and various other phenomena connected with the ascent of sap, are deter-

mined by the relative gain and loss of water by absorption and by transpiration. The problem is highly complex, since the different organs of absorption, of conduction, and of excretion are subjected to different conditions and to diverse modes of stimulation. The root buried in the soil is to a great extent protected from the fluctuating changes in the environment. It may nevertheless exhibit a diurnal periodicity ; though whether or not such periodicity exists is not definitely known, and it would be necessary to undertake an investigation on the subject. I have observed that exposure of the stem to the action of light has the remarkable effect of checking the rate of conduction (p. 47). Sunlight, which by its heating effect enhances the transpiration from the leaves, may thus exert an inhibitory action on the conduction of water in the stem. Finally, the transpiring leaves are sub- jected to the numerous fluctuating changes of the environ- ment, to variations of humidity, to mechanical disturbances caused by the wind, to variation of temperature, and to the alternating action of light and darkness.

The combined effects of these varying influences, which act unequally upon the three regions of the plant, are thus seen to be very numerous, and the complications which thus arise may well appear baffling. But the difficulty in the solution of such intricate problems will by no means be lessened by the employment of mere verbal phrases, nor by any argument of a teleological character which offers no real explanation of the underlying physio- logical mechanism. Nor can any scientific advance be made by the unjustifiable employment of physico-chemical processes in explanation of phenomena which are beyond their scope. There has hardly been any recent contribu- tion to plant-physiology so important as that of the inves- tigation of osmotic action. But it would be a distortion of truth if it were to be assumed that the extremely slow osmotic action could give rise to a velocity of ascent of sap which may be as high as 70 metres per hour ; or that

the quick pulsatory movements of certain plant-organs is brought about by the alternate and spontaneous manu- facture and destruction of osmotically active substances. The problem of the ascent of sap and its diverse mani- festations, though highly complex, is not insoluble. By isolation and separate investigation of the individual factors it will be possible to remove the obscurity which surrounds it. It is obvious how necessary it is to isolate an individual organ for the study of its characteristics. Neglect of this has often led to conclusions which are quite unjustifiable ; thus from the observation that the application of dilute acids at the root induces a reduction in transpiration, it has been concluded that this agent has a retarding effect on transpiration itself. This in- ference is not logical, since the effect might as well have been due to an induced variation in the absorptive power of the root, or in the conducting power of the stem. It is more likely that the chemical agent affected all the different activities of absorption, conduction, and tran- spiration alike.

The difficulty attending the separate investigation of the activities of the three regions of the plant arises principally from the absence of exact methods and suitable apparatus for investigation : but I have endeavoured to overcome it by the invention of various instruments of precision which will be presently described. Complete isolation of the different regions frcm each other is im- possible, since, as will be shown, a physiological continuity exists throughout the plant. Approximate isolation may, however, be secured. Experiments have already been described on the ascent of sap in isolated stems from which the root and transpiring leaves had been removed. The root may be isolated by cutting the root-stock close to the ground and studying its activity by observing the rate of exudation from the cut end of the stock. Finally, the trans- piring activity may be studied by taking a single leaf with the cut end of its short petiole immersed in water.

The current view of transpiration is that it is mainly a phenomenon of physical evaporation. Thus, to quote Pfeif er : ' Transpiration is influenced by the same external conditions as the evaporation of water in general. . . . Transient and rapid changes, such as the movements of the stomata, serve to modify the transpiration according to the conditions existing at the moment, and thus exercise a certain regulatory control, . . . The actual evaporation of water is a purely physical phenomenon, dependent in a plant, as in a dead body, upon the physical properties of the body in question.' ^

The object of the following experiments is to ascertain whether the giving up of water by the transpiring leaf is essentially a physical process, as Pfeffer suggests in the above quotation, or a physiological process of excretion effected by the pulsatory activity of living cells. In carrying out this investigation on transpiration it was necessary to devise a sensitive apparatus by which the rate of normal excretion and its induced variations could be rapidly determined with a high degree of accuracy, which will now be described.

A moderate-sized transpiring leaf is mounted water- tight in a graduated vessel with a side-tube containing a drop of non-adhesive oil to act as a valve. This prevents evaporation from the side-branch, and also serves as a means of counting the air-bubbles that enter the vessel from the outside (fig. 27). Transpiration, by removing a certain quantity of water from the vessel, causes a slight vacuum ; the normal pressure is, however, immediately restored by a bubble of air, which enters the vessel by lifting up the oil-valve. The drop of oil falls back and the valve is closed once more, and the process is repeated time after time.

Each bubble thus indicates the removal of a definite quantity of water by transpiration. This Bubbling Method has proved to be a very accurate and valuable means of investigation. Under constant external conditions the interval between successive bubbles is extremely regu- lar. Thus transpiration from a full-grown leaf of Naiiclea, kept in a room temperature of 30° C, caused the appearance of successive bubbles at exact intervals of ten seconds, without any variation. The specimen was next removed to a cooler room with a temperature of 28° C. The bubbling period now be- came slowed down to twelve seconds, and this remained constant for the next hour. It will presently be shown that this depression in the rate of excretion is due not to physical but to physio- logical variation. Other experiments will be de- scribed which will show that definite physiological changes are attended by induced variation in the ex- cretion, as detected in the change in the bubbling period ; that is to say, a stimulatory agent quickens the rate of bubbling ; depressors, on the other hand, cause a slowing down of the rate. The rate of bubbling observed in a given specimen depends (i) on the sensibility of the apparatus, (2) on the transpiring activity of the species of

the plant, (3) on the size of tlie leaf and (4) on its physio- logical condition. The rate of bubbling, on account of the above circumstances, exhibits a wide variation in different specimens. But in one and the same specimen a great uniformity of excretion is observed under constant external conditions. The absolute rate of transpiration may easily be deter- mined by finding the constant of the apparatus. For this purpose we ascertain, by a sensitive balance, the difference of the weight of the Bubbler containing the leaf at the beginning and at the end of an hour. This difference represents the loss of weight by transpiration. A count having already been taken of the number of bubbles in the course of the hour, each bubble represents the loss of a definite quantity of water by transpiration. Thus, in a particular experiment, the leaf of Thiinbergta grandifiora lost o • 168 gram of water in the course of an hour, during which 60 bubbles were counted. The loss per bubble was thus 0-0028 grm. With a still more sensitive apparatus it is possible to detect a loss of one mgrm. Determination of the loss of water by weighing requires a long time, whereas the Bubbling Method enables us to obtain an immediate indication of the absolute rate of transpiration and its induced variations.

In less vigorous specimens of leaves the average rate is found to be constant, though the intervals between successive bubbles vary slightly above and below the mean interval. Thus in a particular leaf of Thiinhergia the successive bubbles occurred at intervals of 29, 31, 30, 30, and 29 seconds. In order to find the relative transpiring activity of different species of plants, I took leaves the area of which was nearly the same. I thus found that the rate of trans- piration of Thunhergia was half that of Naiiclea. In Crassu- lacese the transpiration is very feeble ; in Bryophyllum calycinum the activity of excretion was found to be one- fifth that of Nauclea. I also determined the evaporation

of water from a free surface, and the transpiration from an approximately equal area of leaf. Representing the evaporation as loo, transpiration from Nauclea was 45, from Thunhergia 20, and from Bryophylhim calyciniim it was 9. Ratio of Transpiration from the Upper and Lower Surfaces of Leaves Many leaves bear stomata only on the lower surface, on account of which transpiration at that surface is relatively greater. This may be qualitatively found by the use of cobalt paper, which becomes reddened earlier at the lower surface. To obtain quantitative results, Garreau errployed the laborious method of cementing the leaf in two bell- jars containing vessels of calcium chloride. The relative increase in weight of the two vessels of calcium chloride gives the amount of water transpired respectively by the upper and the lower surfaces of the leaf. The following method is more direct and simple ; the results moreover are very accurate. The cut end of the petiole of the leaf is fixed air-tight in the apparatus (fig. 27), which is then placed on a sensitive balance, and the loss of weight deter- mined for, say, fifteen minutes ; this is the total transpira- tion, T, for both the upper and the lower surfaces. The upper surface of the leaf is then smeared with freshly boiled vaseline, which prevents transpiration from the upper surface : the loss of weight in fifteen minutes now indicates the transpiration, l, of the lower surface only. The leaf is now smeared on the lower surface as well ; the loss of weight should now be zero ; if any loss occurs in this condition, it must be due to some unavoidable leakage ; in practice this is found to be negligible. Having found the total transpiration t, and l, the transpiration from the lower surface, t— L gives the transpiration from the upper surface. Representing the total transpiration, T, by 100, we thus obtain the percentage of transpiration

at the upper and lower surfaces respectively. The ex- periment is next repeated with a fresh specimen, but this time the lower surface is smeared with vaseline, which gives u, the transpiration from the upper surface : t— u is then the transpiration from the lower surface. The results of the first experiments were not found to be very consistent ; this was traced to impurities in the vaseline, which contained traces of moisture. The difficulty was overcome by boiling the vaseline before applying it to the leaf. With this precaution the results were found to be highly satisfactory The following is a summary of the results.

The leakage is thus seen to be negligible ; the average ratio of transpiration from lower and upper surfaces of the leaf of Nauclea is thus 76-6:22, or about 3*5:1. In Thunhergia the ratio is about 4:1. It may be of interest to obtain an approximate idea of the transpiration from an individual stoma at a temperature of 30° C. By means of a standardised Bubbler, the loss of transpiration from both the surfaces of a particular leaf of Nauclea was found to be 600 mgrm. per hour. The transpiration from the lower surface of the Nauclea leaf

is, as we have seen, H part of the total, and was therefore 467 mgrm. per hour. Portions of the epidermis taken from different parts of the lower surface of the leaf gave the average number of stomata to be 820 per sq. mm. The area of the leaf was 20,800 sq. mm. ; hence the total number of the transpiring stomata was approximately 17 millions. Transpiration from an individual stoma was thus about 0*000028 mgr. per hour. In investigating the induced changes of transpiration, it is not necessary to determine the absolute rate, but only the relative variation. Thus in an experiment on the effect of change of temperature, the normal rate of transpiration was one bubble per ten seconds, or one-tenth of a bubble per second. In order to avoid fractions, it is better to take an hour for the unit of time. The hourly rate of transpiration at 30° C. was thus 360 bubbles, and it was depressed to 300 bubbles per hour at 28° C. The rela- tive change in the transpiring activity induced by slight cooling is thus in the proportion of 360 : 300. As the in- vestigation on the induced variations of activity of trans- piration is carried out with an identical specimen and with the same bubbler, it is sufficient to determine the ratio of the normal rate to that of the changed rate. The trans- piratory activity will therefore be relatively measured by the number of bubbles per hour.

It has already been mentioned that the generally accepted view of transpiration is that it is essentially a phenomenon of physical evaporation. I am, however, able to describe several decisive experiments which prove that excretion from leaves takes place even in the absence of evaporation, thus affording a conclusive proof that transpiration is an active physiological process. The first experiment of the series was carried out in the Mayapuri laboratory in the hill-station of Uarjeeling

during continuous downpour of rain on the break of the monsoon. The air was surcharged with moisture. The tem- perature indoors was i6° C, and the transpiring activity of a single leaf of Hydrangea was found to be 40 bubbles. Water was sprayed on both the upper and lower surfaces of the leaf ; this did not arrest the transpiration ; the bubbling persisted at the rate of 24 per hour. In a second experiment the transpiring activity during a heavy downpour was 38, the leaf being placed outside, but protected from the rain. The transpiration persisted, after exposure to the rain, at the same rate for half an hour, after which it was lowered to 20.

The next experiment is still more decisive ; evaporation from the leaf was prevented by thickly coating both the upper and the lower surfaces of the leaf with freshly boiled vaseline. It is true that the leaf under this abnormal condition is deprived of the supply of oxygen on which the various activities of its life depend. If it be a case of active secretion, this will induce only a depression of the rate, but not arrest. If, on the other hand, transpiration is dependent on evaporation, the fact will be demonstrated by the immediate arrest of transpiration. Experiments on these lines were carried out with various leaves. Thus in a vigorous leaf of Nauclea the normal rate of transpiration was 180 bubbles per hour ; after smearing both the surfaces with vaseline the bubbling was found to persist ; after an hour the rate was 139 ; at the eighth hour the excretion was still persistent, the rate being 30 bubbles per hour. The results of other experiments were similar, the only difference being that in less vigorous specimens the decline of the rate of bubbling was more rapid than in the above case.

It may now be asked : what happened to the excreted water ? Examination of the smeared leaves showed that the excreted water became collected in small patches under the film of vaseline, which prevented its escape into the atmosphere. This was specially marked on the lower surface of the leaf which bore the greater number of stomata. The experiments described above prove conclusively that the excretion from leaves is an active process independent of evaporation.

I shall, in order to avoid ambiguity, use the term trans- piration only in the sense of active excretion in the further discussion of the subject. Though evaporation is not essential for excretion, it is important in the removal of the excreted water, and thus in maintaining a state of diminished turgor in the transpir- ing region. We have seen that the flow of sap is determined by the turgor-gradient, and this difference could not be permanently maintained unless evaporation quickly re- moved the excreted water, and caused a partial drought at the upper end of the plant. The reason for the gradual diminution of excretion in the vaselined leaf is the accu- mulation of water which could find no vent for escape, and which tended to produce a flow of sap in the reverse direction.

The ascent of sap involves physiological continuity throughout the plant. The existence of this continuity is demonstrated in the fact, already described (p. 48), that, in a plant subjected to drought, the leaf on being supplied with water exhibits absorption, thereby pro- ducing a reversal in the direction of the flow of sap. The terminal leaf in the above case functions as a root, that is to say, the organ which excretes is also capable of absorption.

Further experiments are given below which prove the existence of this continuity in the leaf. Just as it has already been shown that the root and the shoot are affected alike by definite physiological changes, so now it will be shown that the petiole, the midrib, and the lamina respond in an identical manner. The effect of raising the temperature of the water at the cut end of the stem, in enhancing the rate of ascent, has already been described, as also the converse effect of a fall of temperature (p. 58). I have now to describe experiments on the effect of thermal variation on trans- piration, (i) when the lamina is subjected to a change of temperature, and (2) when the distant petiole is subjected to thermal variation, the lamina itself being kept at a constant temperature. As regards the effect of change of temperature on the lamina, it may be remembered that the transference of a leaf from a warm to a cold room was found to be attended by depression of the rate of transpiration (p. 85).

The experiment on the effect of variation of the temper- ature of the petiole was carried out as follows. The cut end of the petiole of a leaf of Naiiclea was mounted, with a thermometer, in a metallic tube. This tube was placed inside a larger vessel, which could be filled with water at different temperatures. I first produced a gradual lowering of temperature from 29° C, which was the temperature of the room, to 10° C. It is to be understood that the petiole alone was subjected to the lowering of the temper- ature, the lamina being maintained at the temperature of 29° C. After completion of the series of observations, the temperature inside the tube was allowed to return to the room-temperature, and afterwards raised from 29° C. to 32° C. The following table shows the effect on transpiration of cyclic variation of temperature of the petiole.