Transpiration and the ascent of sap in plants

Preface

The Ascent of Sap is a problem of old standing. As was the case with several other biological problems, a peculiar alternation may be observed in the hypotheses formed to explain the phenomena. At first the process of the ascent of water in trees was, almost without serious thought, assigned to the vital activities of the plant, and, in general with other vital processes, put outside the domain of physical investigation. Later, advances in physics and chemistry, introducing rationality into observation, emboldened hardy spirits to assign the whole process to various physical forces or to combinations of them. Doubtless these philosophers somewhat trans¬ gressed legitimate deduction, and their daring met its punishment in the overthrow of their successive physical theories. It was then again the turn of the Vitalists, and during the latter part of the last century they enjoyed their heyday of dogma. However, the leaven of ration¬ ality still worked on in even their theories, and several physiologists must have felt what Strasburger stated, that the physical forces developed in, and the physical configuration exhibited by, the water tracts would supply a complete explanation when properly understood.

In the present monograph, an account is given of a

VI

PREFACE physical explanation of the rise of water in trees. This theory rests on the knowledge of a property of liquids, which, although discovered in the middle of the last century, was little recognised and seldom referred to in physical literature. It now appears that a full appreci¬ ation of this property is essential for the realisation of the manner in which water is raised in plants and of the meaning of the structure of trees as a mechanism for lifting water.

In the formation of the theory and in much of the earlier work carried out to test its validity and to illustrate its applicability, Dr. J. Joly and the author worked in collaboration. The work on the cryoscopy of sap was carried out with the help of Mr. W. R. G. Atkins, to whom the author is further indebted for his help in reading the proofs and for his useful criticisms. Prof. R. A. Gregory — the editor of this series of monographs — has also kindly supplied several valuable emendations and suggestions.

Henry H. Dixon.

School of Botany,

Trinity College, Dublin.

Chapter I

Transpiration and evaporation. — Comparison of the transpiration from the upper side and of that from the under side of hypostomatous leaves has shown that transpiration is principally effected through the stomata. The total area of the stomata, however, bears but a very small proportion to that of the whole leaf. For example, in a leaf of Cataljpa bignonioides it is but O' 9 per cent, of the total leaf surface. In a leaf of Heliantlnus annuus the area of the stomata appears to be about 3 per cent, of the whole leaf surface.

Many years ago, Unger showed that under similar con¬ ditions the amount of water evaporated from a free surface was 2-8 to 138 times that transpired from an equal leaf surface. If we take the lower limit, this means that 35 sq. cm. of water surface will give off as much water vapour as 100 sq. cm. of leaf surface under similar con¬ ditions. Of this 100 sq. cm. as much as 3 sq. cm. may be represented as stomata. So we arrive at the surprising result that the combined area of the stomata is at least ten times as efficient in giving off water vapour as a free water surface of equal area.

B

Diffusion through stomata. — The beautiful re¬ searches of Brown and Escombe on diffusion through perforated septa have satisfactorily cleared up this diffi¬ culty. They showed experimentally that the amount of vapour which diffuses through a perforation in a septum does not diminish proportionally as the cross section diminishes, but only in proportion as its diameter decreases. This result, which is at first sight by no means obvious, has been mathematically explained by both Stephan and Larmor. Brown and Escombe quote both writers ; but as their methods may not be easily followed by all botanists the following simple explanation may be of interest.

When diffusion, undisturbed by convection currents, is taking place from a perforation which is kept filled with water vapour, or from a surface of water, into a space less rich in water vapour, it is evident that after a time a certain gradient of concentration will establish itself over the aperture. The concentration will be greatest near the perforation and less more remote from it, whilst shells of uniform concentration will be found over-arching the perforation. At a little distance from the aperture, say when the diameter of the shells is about four times that of the perforation, these shells will be approximately hemi¬ spheres.

- To possess any given concentration p a shell must have an area bearing a definite proportion to the area of the perforation ; because the supply maintaining its concen¬ tration is derived from the water molecules leaving the aperture, and the number of these will be proportional to its area.

Let R and A denote the radius and area respectively of the shell having a concentration p, and r and a the radius and area of the perforation. Let p1 indicate the concen¬ tration of water vapour in the perforation.

A — La or It2 = hr2.

I

The flow into the shell = the gradient x area = x A

x A = /i’7r»,2= (Pl-p)Alcnr.

R r sjk

Therefore the flow from the aperture is proportional to its radius and not to its area.

Under the “ static ” conditions to which these calcula¬ tions apply, the shape of the stomata also contributes to their efficiency. It will easily be understood that separated as they are from one another by distances relatively great compared with their diameters, the diffusion of water vapour from adjacent stomata will not interfere. Conse¬ quently the rate of diffusion at the margins will be greater than over the middle of the apertures. Therefore, an opening having the longest margin relatively to its area will be the most efficient ; and the slit-like form of the stomata is seen to be particularly advantageous.

The consideration that the margins are the most effec¬ tive parts of the stomata in diffusion suggests another way of looking at the “ diameter law.” It is evident that for very small holes the marginal region bears a very large relation to the whole opening. For circular apertures the area decreases as the square of the radius while the margin is reduced only as the radius. In the case of slit-like apertures the whole opening may be regarded as marginal. But it is this marginal region which is most effective ; and therefore we should expect the amount of water vapour diffusing through such an aperture to be approximately proportional to the margin (and therefore to the diameter) rather than to the area of the aperture.

The diffusion of water vapour from the intercellular spaces of the leaves through the stomata has been thus explained on simple physical principles. It remains to be seen how the supply of water to these spaces is to be accounted for.

Functions of evaporation, osmosis, and im-

B 2

TRANSPIRATION AND ASCENT OF SAP ch.

bibition in transpiration— The leaf tracheae contain¬ ing the rising sap are separated from the intercellular spaces by a layer of one or more thin-walled cells. The thin wall, which is formed of cellulose, is permeable to water and dissolved substances ; at the same time it is tough and of considerable tensile strength. It is lined by a layer of viscid protoplasm which in turn surrounds, and is completely filled by, a solution — the vacuole. The latter contains various carbohydrates and electrolytes in solution, and round it the protoplasmic layer forms a fairly perfect semi-permeable membrane. It is evident that, when sufficient water is available, the solutes in the vacuole will exert an osmotic pressure on the protoplasm which will be forced outwards against the wall and distend the latter. Ultimately, the tensile stress in the wall will balance the osmotic pressure of the solutes of the vacuole and equilibrium will be established and the cells will be tense and rigid. The walls of the tracheae are also quite permeable, but they are more rigid than those of the cells and they are further prevented from collapsing by the presence of the internal supports in the form of rings and spirals. The tracheae do not enclose a semi-permeable membrane of protoplasm.

We may now consider how this mechanism will act in the transference of water. The imbibitional or capillary forces of the cell-walls in contact with the intercellular spaces of the leaf will draw off water from their vacuoles through the protoplasm until the concentration of solutes in the vacuole is such that the vapour pressure of water in it is equal to that obtaining in the cell-wall. Supposing now that the vapour pressure of the water menisci in the reseau of the cell- wall is greater than that obtaining in the intercellular space, water will leave the wall and the menisci will retreat into it. This will cause their curvature to increase and will raise their capillary forces so that they will endeavour to extract water from the solution in the

I vacuole. A concentration of tire vacuole results, and con¬ sequently the osmotic pull on the water in the tracheae is increased. Hence it follows that a transference of water from the tracheae will take place so long as the vapour pressure of water in the tracheae is greater than that in the intercellular spaces of the leaf.

With such a mechanism before our minds we can easily realise that the whole process of transpiration may be purely physical and one into which vital actions (viz., actions connected with living substance the intermediate steps of which are little understood) need not enter.

These considerations also explain several observations otherwise difficult of interpretation. When colouring materials (e.g., eosin) or other substances, poisonous or non-poisonous (e.g„ copper sulphate, picric acid, tartaric acid, sodium bicarbonate, ferricyanide of potassium, etc.) are supplied in watery solution to the cut surface of a transpiring branch, it is found that the rate of transpira¬ tion continues without much diminution till the solution can be detected in the leaves, but then suddenly falls off. This may be seen either by direct measurement of the water drawn up, or by the fact that the leaves of these branches usually remain fresh until the arrival of the solution in them, but then rapidly lose their turgor, flag, and finally become dry and crisp. These observations are easily explained if we may assume that at first the solu¬ tion is drawn up in the conduits under the tension set up through the osmotic forces of the cells of the leaves, but afterwards, when the upper portion of comparatively pure water has been eliminated from the plant by transpiration, the solution comes into contact with the living cells and modifies or destroys their osmotic properties. The latter soon lose their turgor, and the leaf droops.

That the loss of turgescence will necessarily be attended by a diminution of the amount of water evaporated from the leaf will appear evident when it is considered that :

First the transpiring surface of the evaporating cells will be diminished owing to the contraction of the cells when they cease to be turgescent. Secondly, the diffusion of the water-vapour from the inner tissues of the leaf will be hindered by the collapse of the intercellular passages caused by the drooping of the leaf ; and, finally, the evaporating films which on the surface of the wall of the turgid cell may be supposed to be but slightly concave, will under the new conditions tend, by retreating inwards into the sub¬ stance of the cell-walls, to form a series of more concave menisci from which, as is known, evaporation will proceed more slowly. However, as the walls of these cells are im¬ bibed with water, and a continuous column of water extends back from the outer evaporating walls down through the conduits into the plant hanging from the surface film formed on the outside of the evaporating cell-wall, a slow movement upwards will take place of the solution in the conduits even after the loss of turgor by the osmotic cells. The supply is in most cases inadequate to prevent the drying of the leaf.

To the evaporation at the surface film formed on the cell- walls must also be referred the ascent of water in pieces of dead wood which have been soaked and injected with water, as described by Strasburger ; for in this case, of course, the intervention of osmotic forces is excluded. It is to be noticed that in this experiment also the wood dries downwards from above.

From the foregoing considerations it seems quite feasible to explain the process of transpiration as a purely physical one in which the intervention of living matter, as such, is not necessary ; for we might substitute a dead osmotic cell in the plant for the living one without having reason to expect a difference in the action of the mechanism. This being so, it appears highly probable that transpira¬ tion is often a purely physical phenomenon.

However, we must not shut our eyes to the fact that i several observations seem to support the view that under normal conditions some sort of vital action intervenes.

Transpiration of living and dead leaves. — It was noted above that when the leaves of a branch are killed, not only is the flow of water upwards greatly reduced, from causes which have been already explained, but ultimately the leaves dry up and finally fail entirely to raise water in the branch. It is true that in this case, not only are vital actions removed, but also one of the most important features of the mechanism, viz., the semipermeable membrane, is destroyed by the coagulation of the protoplasm. It is evident, however, that after this the capillary forces of the cell-wall of the leaf-cells alone are unable to continue to raise the water under the new con¬ ditions, and this would suggest that unaided they may be insufficient in the living leaf.

This line of reasoning would indicate that the protoplasm may not only act as a semi-permeable membrane by allow¬ ing water to pass through to a region of diminished pressure, but it may also actively secrete water, or a solution, on its outer surface. Such glandular action of protoplasm is well known, and examples from the coenocytic fungi may serve as illustration. In Phycomyces, Pilobolus, and Mucor, water which is absorbed by the submerged part of the coenocyte is expelled on its aerial surface. The same process may be witnessed in the nectaries and all the glands of higher plants.

In these cases there is some reason to believe that the liquid exuded is not pure water ; and hence it seems pro¬ bable that the protoplasm first secretes soluble substances on its outer surface and that these act osmotically when concentrated by evaporation, on the vacuoles within and draw water from the cells. Where the process is very rapid, as, for example, the expulsion of water from the cells of the pulvinus of Mimosa in response to a stimulus, such a sequence of events can scarcely be imagined. The extremely rapid action seems only explicable by assuming some sudden change in the permeability of the proto¬ plasm.

Secretion of water. — The active secretion of water by cells is particularly well illustrated by the water glands in the tips of the leaves of Colocasia antiquorum. As is well known, when this aroid is under suitable conditions of moist soil, saturated atmosphere, and favourable temperature, a succession of drops (often as many as two per second) may be seen issuing from its leaf tip. A similar exudation of fluid may be often observed on the leaves of grass-seedlings when surrounded with a satu¬ rated atmosphere.

It is evident that this exudation of fluid would be easily explicable on purely physical processes if the fluid con¬ tained any considerable quantity of dissolved substances. Then the external solutes might be assumed by virtue of their osmotic pressure to extract water from within and so keep up the supplies on the outside of the cell. The pres¬ ence of dissolved substances may be sometimes demon¬ strated in the exudations of the fungi by evaporating drops of the fluid on polished glass. How far these dis¬ solved substances are crystalloids, and so competent to act osmotically, remains to be determined. At the same time, it should be noted that, even admitting there are sufficient crystalloidal solutes on the surface of the cell to account for the exudation, it seems we must assume a secretory action in the protoplasm to bring these crystalloids out of the cells and expose them on the outer surface, and this action must be a continued one as the exudation will constantly be carrying off its solutes.

If, however, the fluid exuded is pure water, or contains practically no dissolved crystalloids, it is evident that osmosis cannot account for the process, for the osmotic action of the solutes within the vacuoles cannot cause the elimination of water on the outside of the cells. In this,

I energy is being expended, and the energy apparently must be supplied by the metabolism of the protoplasm.

Fortunately the quantities exuded from the leaf tips of Colocasia are quite large enough to allow us carefully to test the purity of the water. With this intent some 10 c.c. were collected from the leaves of this plant during one night ; and, by means of the thermo-electric method of cryoscopy, which will be described later, were tested with regard to their freezing-point. It was found not to differ sensibly from that of distilled water. The electrical con¬ ductivity was also determined for the sample, and it was found to be less than that of tap water. These tests show that osmotically we may regard the exudation as pure water, and, consequently, the process must be one of secre¬ tion involving the. intervention of living protoplasm and the expenditure of stored energy.

In the case of Colocasia the dripping ceases when the leaves are surrounded with an unsaturated atmosphere. Evidently the secretion is no more than able to keep pace with the demand of evaporation, but at the same time it seems reasonable to assume that the water has been largely supplied for evaporation by a secretory process. It seems also reasonable to suppose that when evaporation is acceler¬ ated beyond the capacities of protoplasm for secretion the cell- walls will dry and the capillary forces of the menisci in their substance will lend their aid in separating the solvent from the solutions in the vacuoles.

How far can we transfer the glandular functions of the leaf tip of Colocasia to the mesophyll cells of the trans¬ piring leaves ? An answer to this question has been sought by different ways.

Transpiration into various gases. — If the cells of the mesophyll secrete pure water on their outer side we should be justified in expecting that this secretion, in common with other vital actions, would be inhibited when the protoplasm was anaesthetised, or would be io TRANSPIRATION AND ASCENT OF SAP ch.

reduced when its activities were depressed. Starting from this idea an endeavour was made to test the effect on transpiration of an application of chloroform vapour, ether vapour, and carbon dioxide gas. Although each of these three agents, when they were brought round the transpiring leaves, led to a marked falling off in the amount transpired, consideration showed that their actions are so manifold that the effect observed cannot with certainty be attributed entirely to their direct action in checking the vital actions of the protoplasm. At the same time, experi¬ ments were made to test the effect of an increased supply of oxygen on transpiration ; the marked acceleration pro-

Fig. l.

duced by this gas possibly is due to its stimulating effect on the secretion, but in any case is of considerable interest in this discussion.

The method of experiment was as follows : The rate of transpiration of a branch enclosed in a large receiver, and supplied with a constant current of dried air, was observed. This rate was then compared with the rate of transpiration, when a similar current of some other dried gas, or dried air, carrying with it some anaesthetising vapour, was passed through the receiver.

The rate of transpiration was estimated, either by the motion of an index moving in a capillary tube sealed i THE NATURE OF TRANSPIRATION n

hermetically to the cut end of the branch, or by directly weighing the amount of water transpired. In the latter case, which was found to be the more satisfactory, the branch, inserted through a caoutchouc cork into a test-tube containing water, was hung from one arm of a balance. The arrangement is shown in Fig. 1. In this figure b is a tower ’ containing calcium chloride, and c is a sulphuric acid bulb for drying the gas supplied. Before passing through the drying materials, the gas enter¬ ing at e is led into an inverted flask d, which is provided, in addition to the tubes of entry and exit, with a U -tube, /, filled with oil. The supply of gas is adjusted until the oil in the longer arm of the tube / is brought to a certain level. By this means the pressure, and consequently the flow, of gas through the apparatus, can be adjusted and compared. When vapours are to be supplied, the liquid from which the vapour is derived is placed in a sulphuric acid bulb, like that in the figure, but inserted in the train between the air supply and the flask d. In each case the supply was passed into the apparatus from a cylinder containing the compressed gas.

The chamber containing the branch was exposed only to a very feeble light so that the stomata were closed throughout the experiments.

The first experiments made were with the index method of estimating the rate of transpiration. A modification of the apparatus, as figured, which is readily understood, was then used, and the branch, sealed hermetically to a capillary tube containing the index, was inserted from below into the receiver.

With these arrangements there soon appeared to be a marked difference in the rate of transpiration in oxygen and carbon dioxide. Thus, to quote the mean of a number of observations with a branch of Cytisus laburnum in carbon dioxide the index moved 1 cm. in 38 sec. ; with the same branch in oxygen it moved 1 cm. in 28 sec.

When the rate of transpiration in air was compared with that in oxygen, it was found that when a branch was surrounded with the latter gas, transpiration was slightly more rapid. The index for the branch in air moved 1 cm. in 36 sec. ; for the same branch in oxygen it moved 1 cm. in 33 sec.

If, while a current of air was passing through the receiver, a piece of cotton wool soaked in chloroform was intro¬ duced, a much more marked difference in the rate of transpiration became apparent in a short time. Thus the index was traversing 10 cm. in 50'8 sec. before chloroform was introduced. Thirty minutes later the index took 516 sec. to traverse the same distance. The chloroform was then removed, and the air current maintained for 60 minutes. At the end of this time the index moved 10 cm. in 120 sec. This result is the mean of a number of experiments made with a small branch of Acer macrophyllum.

With another branch in air the motion of the index was 10 cm. in 127 sec. When surrounded with chloroform vapour for 45 minutes the index took 642 sec. to traverse the same distance.

A similar diminution in the rate of transpiration is observed when the branch is surrounded by ether vapour. Thus, with a branch of Acer macrophyllum in air, the index moved 10 cm. in 205 sec. ; with the same in ether vapour it moved 10 cm. in 265 sec.

These experiments indicate a large difference in the rate of transpiration in the different gases. The figures given here will serve only as examples of the results of such experiments, for, although they were the means of a number of observations, the latter are made so precarious by various circumstances, that they can only be taken as indicating a difference, and not as giving a measure of it. The sticking of the index in the capillary tube, and the opening of the receiver to introduce the anaesthetics, bring i THE NATURE OF TRANSPIRATION 13

in errors, which render the method nnsnited to exact observation.

In order to eliminate these sources of inexactness, recourse was made to the arrangements shown in the figure. The results obtained by this method are displayed in the following table. The difficulties of keeping the flow 1 of gas exactly constant through the apparatus, and other experimental errors, lead to variations between the indi¬ vidual observations often amounting to 10 per cent. ; but by multiplying these observations, an approximation to the actual alteration in the rate of transpiration has been obtained. The numbers here given are the means of a large number of observations. In each case, the branches experimented on were from a bush of Syringa vulgaris, except in the experiments where effect of ether vapour was observed. In these, branches of Cytisus laburnum were used.

If the amount transpired in air be taken as 100, the amounts transpired in the other gases are as follows. These figures may be said to denote the specific transpiration for the gases

Medium.

Specific

Transpiration.

Oxygen .

135-8

Air .

100-0

Carbon dioxide .

87-3

Ether .

82-3

Chloroform .

06-4

The first source of error affecting these experiments, and one which it seems hard to eliminate, arises from the fact that the effects of the different gases may be more or less rapid. Thus it is very certain that the light gases

1 It is to be observed the rate of flow of the different gases will be different, even if the pressure be the same.

TRANSPIRATION AND ASCENT OF SAP ch.

will diffuse into the intercellular spaces more quickly than the heavy gases, and so come into contact with evapora¬ ting cells more quickly. Besides this, it seems probable that the poisoning and anaesthetic effects of one may be more rapid than those of another. The observations, on which the numbers given above are based, were com¬ menced in each case after the branch had been surrounded by the gas for five minutes, and were discontinued before any lethal effects could be observed in the leaves ; for such, if arising, would cause the osmotic pressures obtaining in the leaf-cells to become diminished, by rendering the proto¬ plasmic membranes permeable. These effects were usually visible within 45 minutes after starting the experiment. It is possible that the denser vapour could not, within this time, diffuse into all the intercellular spaces of the leaves.

An error arising from this possibility is most unsatis¬ factory, as it seems extremely difficult to make proper allowance for it. It seems impossible, at present, to decide how soon the surrounding gas will come into con¬ tact with the evaporating cells, and, also, when the anaesthetising or stimulating action will cease, and the lethal effects will begin, if, indeed, there is any sharp line of distinction.

Next we come to an error which can, in some degree, be eliminated. It is known that the rate of diffusion of a gas will be influenced by the nature of the gas occupying the space into which it is diffusing. Thus water-vapour will diffuse more slowly into carbon dioxide gas than into oxygen. This difference depends on the relative sizes of the molecules of the gases into which the water-vapour has to diffuse. For the same pressure and temperature, there will be the same number of molecules of these gases in the surrounding space ; but if their sizes are different, it is plain that the water-molecules will less readily diffuse into the space occupied by the gas composed of the larger molecules.

I

In order to form some idea of this effect, I suspended a shallow dish containing water in the receiver, previously occupied by the transpiring branch ; and in connection with the train of apparatus previously described, successive weighings gave approximately the loss of water by evapora¬ tion from this dish. During the experiment a stream of gas, dried as before described, was kept up through the apparatus. The rate of evaporation, when this current was composed of air, oxygen, carbon dioxide, and largely of ether and chloroform, was observed.

Again denoting the loss of weight of a vessel of water in air as 100, the loss in the other gases was found to be as follows

Medium.

Specific

Evaporation.

Oxygen .

Air .

Carbon dioxide ....

Ether .

Chloroform .

From these observations it would appear that the rate of transpiration is diminished when the leaves are sur¬ rounded by carbon dioxide, ether vapour, or chloroform, much in the same degree as the rate of evaporation would be diminished by the presence of these gases ; and this diminution is in the inverse order of their densities. In the case of oxygen, however, the rate of transpiration is increased much more than the rate of evaporation would be from a liquid surface.

It must be understood that these numbers only apply to the first effects of carbon dioxide, ether, and chloro¬ form; for when these gases begin to exercise a lethal action on the cells, the rate of transpiration is very markedly diminished, presumably owing to the reduction of the osmotic pressure in the cells.