The Physiology of the Ascent of Sap

physiological. I describe in the succeeding chapters several appliances of great sensitiveness which I have been able to devise for the purpose. Returning to the physiological theory, it should be borne in mind that a vague assumption of protoplasmic activity is not a sufficient explanation of the phenomenon of the propulsion of sap in plants. It is necessary further to determine the character of the cellular activity underlying the ascent, how that activity is initiated, and by what means a definitely directed transport of sap is maintained.

As regards the last point, no satisfactory explanation has been offered. Still greater difficulties and complica- tions are introduced when we take irito account other phenomena connected with the ascent of sap, such as the root-pressure, the occurrence of positive and of negative pressure, and the relation between the root-pressure and ' bleeding ' of injured plants. The root-pressure is sup- posed to force the water up and thus to help in the ascent of sap. But when this pressure is most needed, as during the rapid ascent of water to meet active transpiration by the leaves, it disappears or becomes nega- tive. The internal pressure of the tree is also subject to changes which appear to be erratic. Finally, the pheno- menon of ' bleeding ' is supposed to be due to root-pressure. No definite relation is however found to exist between the pressure and the exudation at the cut surface ; the Palms, in fact, exhibit vigorous exudation in the complete absence of any root-pressure.

It is thus seen how necessary it is to arrive at a compre- hensive theory which will explain not only the ascent of sap but also other phenomena associated with it, which are quite inexplicable in the existing state of our knowledge. My object in the present work is to attempt to formulate such a comprehensive theory, based upon experimental evidence. Reference may be made to a long course of investigation which 1 undertook (1904-1906) on the subject of the ascent

of sap.^ It was shown that the transport of water is main- tained by physiological action, and that it is not the mere presence of living cells, hut their rhythmic or pulsating activity, which maintains the ascent of sap. Very little definite information has hitherto been available as regards the characteristics of the rhythmic vegetable tissues. A detailed account of investigations on the subject will be found in the works just referred to : but I give in the next chapter a brief statement of the characteristics which distinguish the pulsating from the ordinary tissue, for these criteria will afterwards be em- ployed in proof of the pulsatory character of the tissue concerned in the ascent of sap.

Rhythmic vegetable tissue — Autonomous pulsation in Desmodium gyrans — Multiple response under strong stimulus — Pulsations in growth — Characteristics of pulsatory activity — Effect of variation of internal hydrostatic pressure— Effect of maximal stimulus — Effect of sub-minimal stimulus — Modification of response in sub tonic speci- mens— Effect of variation of temperature on rhythmic activity — Arrest of pulsation at the critical thermometric minimum^ — Effect of anaesthetics — Effect of dose — Action of poison — Tests for pulsatory activity — Summary.

Before describing the characteristics of pulsating tissues in plants, it will be of interest to form a mental picture of the physiological mechanism in the propulsion of sap. I have, in my previous works,^ shown the fundamental similarity of response in plant and in animal tissues. There is in fact no physiological action in the animal which is not to be found also in the plant. This being so, it may be instructive to refer to the means by which one-directioned propulsion of fluids is maintained by animal tissues. Let us take the instance of a multiciliated tissue ; here the cilium at one end gives, as it were, a signal which is followed serially by the rest, the multiple activity being continued for a long time. It is clear that if such a multi- ciliated tissue took the form of a hollow tube, the ciliated surface inwards, and if the tube were filled with water, then, owing to this peculiarity of the multiple-responding cilia, water would be driven in one direction. In the cir- culation of blood in animals, it is the sinus which gives

the signal, and the rhythmic contraction of the heart proceeds towards the ventricle ; the pumping action thus initiated determines the uni-directional flow of blood. Have we any proof that plant-cells are possessed of a similar rhythmic activity ? The detection of this in a single cell is surrounded with many difficulties. Micro- scopical examination, even if practicable, would show little or no effect : for, assuming the diameter of a cell to be of the order of 0-05 mm., its contraction or expansion would not cause any change of more than ten per cent. ; the variation of length would, theiefore, be something like 0-005 mm. The period of a single pulsation of a plant is comparatively slow, being of the order of a minute or so. The problem then is the detection of a rate of change in length of the order of 0-00002 mm. per second, which is beyond the power of a microscope.^

Fortunately we have other means for the detection of rhythmic activity in plants, specially in pulvinated organs. The most striking example of this is found in the lateral leaflets of the Telegraph Plant, Desmodiiim gyrans. The cells of the lower and upper halves of the pulvinule execute alternate contractions : the result of the contraction of the more excitable lower half is a quick down-movement of the leaflet ; while the lower half is in the phase of recovery, the less excitable upper half undergoes contraction with a resulting slow up-movement. The period of a com- plete pulsation varies according to circumstances from a minute to four minutes or so. These pulsatory movements take place without any immediate external stimulus and are therefore described as ' spontaneous ' or self-originated ; such spontaneous pulsation of the vegetable tissue exhibits

^ It is, however, possible to detect ultra-microscopic movements by a special electric method which will be described in a subsequent chapter. all the characteristics of the spontaneous movement of the animal heart.^ There are other plants which exhibit multiple pulsa- tion under special conditions. An example of this is furnished by the leaflets of Biophytiim sensitivum, which are normally in a state of quiescence. Multiple responses of the leaflets are, however, evoked by the application of a strong stimulus, the persistence of the pulsatory activity being dependent on the intensity and duration of the stimulus.

It i€ thus seen that under normal conditions certain tissues, like those of Desmodium, exhibit very pronounced pulsatory activity. In other words, rhythmic activity is strongly developed in certain tissues, while it is but feebly developed in others. The former maintain their rhythmic activity under normal conditions ; whereas intense stimu- lation is required to arouse the latter. Further, there is no strict line of demarcation between the phenomena of multiple and of autonomous response. In very favourable circumstances for absorption of excess energy from without, Biophytum becomes an automatically responding plant like Desmodium. Conversely, under un- favourable conditions, that is to say, when the sum-total of its energy is below par, an automatically responding plant like Desmodium ceases to exhibit any pulsations : but the leaflets, now at standstill, will, like those of Biophytum, give multiple response under strong stimulus.

With regard to the uni-directioned propulsion of fluid in the plant, it has been pointed out that in the animal it is determined by the propagation of excitatory waves. Such a propagation of excitatory waves in vegetable tissues is exhibited in a very striking manner by Biophytum. If we apply a drop of strong salt solution at the inner end of the petiole, repeated excitations will be found to be 1 The pulsatory activity of tissues has been variously described as spontaneous, rhythmic or autonomous, and I use these terms in that sense.

propagated from the point of irritation, in strict sequence, from each pair of leaflets outwards to the next (fig. i). Fig. I. Leaves of Biophytum (left) and of Desmodium gyrans Application of salt at s gives rise to multiple excitation in the leaflet of Biophytum. The lateral leaflets of Desmodium gyrans (right) execute autonomous pulsations. Autonomous activity of a pulsatory nature is also well marked in growing organs,^ as is demonstrated in the records obtained by means of the High Magnification

Fig. 2. Record of Pulsation of Growth taken with the High Magnification Crescograph Crescograph. The growth-pulsations consist of a scries of alternate expansions and contractions, the latter being the smaller of the two ; the resultant growth in length is the difference between the elongations and the contractions (fig. 2). Sometimes the growth-activity alternates on two sides of the organ as the result of lateral pulsations, just as the up-and-down oscillation of the leaflet of Des- modium is produced by the alternate activity of the upper and lower sides of its motile organ.

In previous investigations of these two typical instances of pulsatory activity it has been ascertained that it can be modified in very definite directions by variations of the physiological conditions : a brief summary of these in- vestigations is given in the following pages. If now the ascent of sap be found to be similarly affected by the same physiological variations, it may reasonably be concluded that it too is essentially a phenomenon of pulsatory activity. The following table summarises what has been determined for the movements of Desmodutm and for the rate of giowth, as well as what may be anticipated for the as.cent of sap.

Table I. — Different Modes of Response to Induced \'ariation OF Autonomous Activity dium amplitude, or both Movement of growth j Increased rate of growth Ascent of sap Enhanced rate of move- ment of sap The Characteristic Modifications of Pulsatory Activity under Physiological Variations The effects of physiological variation will be considered in the following order : (i) the effect of variation of internal hydrostatic pressure ; (2) the effect of external stimulus of sub-minimal and of maximal intensity ; (3) the modifying influence of the tonic condition ; (4) the effect of variation of temperature ; (5) the determination of the critical point of thermometric minimum for the arrest of response ; (6) the effect of anaesthetics, and (7) the effect of poison.

(i) The Effect of Variation of Internal Hydrostatic Pressure A certain amount of internal pressure is necessary for the initiation and maintenance of rhythmic activity. This is seen in the renewal of the pulsation in the quiescent heart of the snail when the intracardiac pressure is increased. When a plant is subjected to drought, the turgor and the internal hydrostatic pressure become diminished. A diminution may also be produced artificially by the plas- molytic withdrawal of water. Conversely, an increase of internal hydrostatic pressure may be produced by fixing the cut end of the stem or of the petiole in the short arm of an U tube, and applying hydrostatic pressure by a water column in the longer arm of the tube.

Desmodium Pulsation. — When water is withheld from Desmodium, the leaflet ceases to pulsate, the activity being renewed on irrigation. The arrested pulsation of a de- tached leaflet may also be revived by the application of hydrostatic pressure. The pulsatory activity is thus dependent on the internal pressure. The converse is demonstrated by the plasmolytic withdrawal of water inducing an arrest of the normal pulsation. A solution of KNO3 applied at the cut end of the petiole bearing the pulsating leaflet, induces a continuous diminution of the amplitude of pulsation culminating in an arrest. Restoration of the normal pressure by substitution of water renews the pulsation (fig. 3).

Growth.— FsiTaWel effects are seen in the phenomenon of growth. Growth becomes arrested under drought and is renewed after irrigation. Partial drought diminishes the rate of growth ; application of warm water at the root increases the turgor of the plant and enhances the rate of growth. A plasmolytic solution, on the other hand, diminishes the rate. Thus in a series of experiments with a growing specimen of the flower-stalk of Zephyranthes, the normal growth-rate under partial drought was 0-04 /i

per second. On irrigation with warm water the rate was enhanced to 0-20 yu. ; after this temporary increase the steady growth settled down to o-o8 ^l. On apphcation Fig. 3. Arrest of Pulsation of Desmodium Leaflet due to Diminished Internal Hydrostatic Pressure induced by KNO3 Solution applied at Arrow ; Subsequent Revival on Substitution of Water at Inverted Arrow of KNO3 solution to the root, the rate of growth was found to be diminished to 0-03 /i per second, or to a third of the previous rate.

-Effect of Variation of Internal Hydrostatic Pressure on Growth [Zephyranthes) After application of warm water . Steady growth after one hour After apphcation of KNO3 solution In vigorous specimens, all modes of maximal stimula- tion induce a diminution of turgor, a contraction, and a decrease of pulsating activity. These may be regarded as the normal responses of the plant to stimulus. Desmodium Pulsation. — The inhibiting action of stimulus on the pulsation is seen in the record (fig. 4) of the effect of electric stimulus of moderate intensity. The pulsation is seen to become arrested. On the stoppage of stimulus, the after-effect is often found to be an enhancement above the normal.

Growth. — Various stimuli, mechanical, electric, or photic, retard the normal rate of growth. This retardation in- creases with the intensity and duration of the stimulus, and Fig. 4. Effect of Electric Stimulus on Pulsation of Desmodium gyrans Note inhibition as the direct and enhancement as the after-effect of stimulus apphed at s. culminates in an arrest of growth. Thus under electric stimulation, the normal rate of growth in a specimen was found depressed from o -30 //- to o -09 /* per second. Stronger stimulus induced an arrest. Under the action of light, the rate of growth in a second specimen was found to be diminished from 0-47 fi to o-io /x per second. Stronger intensity of light induced an arrest of growth.

The above results are obtained with maximum stimulus ; sub-minimal stimulus, however, is often found to induce an effect which is opposite to that of the maximal, that is to say, an enhancement of activity. I will now refer briefly to certain very unexpected results obtained in the course of my investigations on the response of vegetable tissues to external stimulus. It was found that the normal sign of response is liable to modifica- tion, the variation being definitely related to the physio- logical condition of the tissue, which may be at or helow par. These two conditions will be designated as the normal and the sub-tonic. This difference in the initial condition of the tissue, though outwardly indistinguishable, is revealed through characteristic changes in the response to a testing stimulus.

The generalisation arrived at in regard to the charac- teristics of response in the two conditions is that, the response of a suh-tonic tissue is of opposite sign to that of the normal. This applies to all tissues, ordinary or rhythmic. As an illustration, the pulvinus of Mimosa normally responds to stimulus by contraction and the resulting fall of the leaf. But if the plant be kept in dark- ness or in other unfavourable conditions, its physiological tone falls below par, and the sign of response undergoes a reversal ; the pulvinus now responds to the same stimu- lus by expansion, and consequent erection of the leaf. Successive stimulations, however, improve the tonic condition, with the result that the abnormal response is gradually converted to the normal. ^

As regards the autonomous rhythmic tissues, their activity declines or becomes finally arrested with increasing sub-tonicity. This condition may be artificially induced by keeping the whole plant or a cut specimen under unfavour- able conditions. The rhythmic activity manifested in pulsation or in growth may thus be made to undergo a continuous decline culminating in arrest. Just as the response of Mimosa in a sub-tonic condition exhibits a response of opposite sign to the normal, so a

rhythmic tissue also exhibits this reversal in sign of response when it is in a sub-tonic condition ; that is to say, that a stimulus which inhibits the activity in a normal specimen, renews or enhances the activity in a specimen which is in a condition of sub-tonicity. Desmodiiim Pulsation. — If a cut specimen of Desmodimn be kept in the dark, the amplitude of pulsation of the leaflets is greatly reduced in the course of about eight hours, and comes to a total stop in the course of eighteen hours. If we now apply the stimulus of an electric shock, the pulsa- tory activity is found to be revived, the persistence depcnd-

FiG. 5. Effect of^Stimulus in renewing Pulsation of Desmodiiim gyrans, originally at standstill Successive exposures to light for five, ten and forty-five minutes. ing on the intensity and duration of stimulation. Similar effects are produced by the application of the stimulus of light. Thus the application of strong light for five minutes gave rise to a single pulsation in a leaflet previously at a standstill. The next application of light of the same intensity was for ten minutes, and this gave rise to four pulsations — two during and two after application (fig. 5). Light was next applied for forty-five minutes, and the pulsatory activity persisted for nearly an hour after the cessation of exposure. These results show that the spon- taneous pulsation, so called, is not self-originated, but is really due to an antecedent external stimulus. The per- sistence of autonomous activity is thus dependent on the amount of stimulation to which the plant had previously

been subjected : the energy supplied by the environment becomes, as it were, latent in the plant, increasing its power of work. Growth. — Diametrically opposite effects of stimulus on normal and on subtonic specimens are also met with in the phenomenon of growth. Thus while the effect of stimulus on a normal specimen is a retardation, its effect on a sub-tonic specimen is an enhancement of the rate of growth. The following table gives the quantitative results of the effect of stimulus on the growth of sub-tonic specimens.

Table III. — ^Acceleration of Growth under Stimulus in Sub-tonic Specimens In the sub-tonic Wheat-seedling, stimulus enhanced the rate two and a half times. In 5. Kysoor also stimulus enhanced the rate of growth by more than thirty per cent. It is thus seen that the effect of stimulus is modified by the physiological condition of the tissue. If we take the cardiac muscle of the animal as an example of rhythmic tissue, it is found that a rise of tem- perature quickens the pulsation ; lowering of temperature, on the other hand, slows it down.

Desmodium Pulsation. — A rise of temperature induces an enhanced frequency of pulsation. Thus in a particular series of experiments it was found that, during a period of twelve minutes, there were four pulsations at 28° C, which increased to six at 31° C, and to ten pulsations at 34° C. growth is similar to the above. It is found that a rise of temperature enhances the rate of growth up to an optimum point which varies in different species of plants. The follow- ing table shows that the rate was continuously increased from 0-03 fjb to 0-92 fi as the temperature was raised from 26° C. to the optimum temperature of 34° C. When the temperature was raised one degree above this optimum point, the rate of growth underwent a decline to o -84 /x.

Lowering of temperature slows down the pulsation of Desmodmm leaflet till at a critical point it becomes arrested. This arrest is however not permanent, since a revival takes place as soon as the temperature is raised above the critical point. As I give in a subsequent chapter records of the arrest and revival of the pulsatory activity below and above the critical point. I need only state here that in Desmodium the pulsation generall}^ becomes arrested at or about 17° C.