ON THE UPTAKE OF SUCROSE AND WATER BY FLOATING LEAF DISKS UNDER AEROBIC AND ANAEROBIC CONDITIONS

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1 [ 13 ] ON THE UPTAKE OF SUCROSE AND WATER BY FLOATING LEAF DISKS UNDER AEROBIC AND ANAEROBIC CONDITIONS BY P. E. WEATHERLEY Department of Botany, University of Nottingham {Received 5 February 1954) (With 7 figures in the text) INTRODUCTION It has long been known from feeding experiments that pieces of leaf may absorb sugar from solutions on which they are floated. Past work on this subject has been reviewed briefly in a previous paper (Weatherley, 1954) in which measurements of sugar uptake by a number of species were reported, together with preliminary investigations on the nature of the uptake. It was suggested that sugar uptake by floating leaf disks is probably an active transfer rather than a passive absorption. If the transference of sugar from the external solution into the cells is an active, energy-requiring process, it would be expected that uptake would be reduced or stopped under anaerobic conditions. This present paper is devoted to a comparison of sugar uptake in aerobic with that in anaerobic conditions, and such a dependence of sucrose uptake on aerobic conditions is demonstrated. Attention is also paid to the changes in water-content of the leaf disks accompanying the uptake of sugar, and estimates made of the permeability constants of the tissues to water and sucrose. The data obtained are consistent with the hypothesis of a passive absorption of water following an active uptake of sugar. The experimental work was carried out at the Universities of Manchester and Nottingham. TECHNIQUE The uptake of sugar was measured as the increase in dry weight of samples of 30 to 40 leaf disks after floating on sugar solution. The details of this method, together with the procedure for collecting the samples, estimating the errors of sampling and treatment of the samples have been given in a previous paper (Weatherley, 1954). As in that work attention was restricted largely to leaf disks of Atropa belladonna and to the uptake of sucrose, but in addition the water-content of the disks was measured simply by subtracting the dry weight of a sample from its fresh weight. To attain anaerobic conditions Petri dishes, containing the floating disks, were placed inside desiccators, each of which could accommodate two dishes. These desiccators contained about 200 ml. of alkaline pyrogallol and were fitted with inlet and outlet tubes so that after the Petri dishes had been placed inside, nitrogen could be blown through them to displace the air. The nitrogen, of chemically pure grade, was passed through a wash bottle also containing alkaline pyrogallol before entering the desiccators. After the air had been 'washed' out of the desiccators, they were clipped off and allowed to stand for

2 14 P- E. WEATHERLEY the period of the experiment without further passage of nitrogen. For aerobic conditions similar desiccators were used each containing 200 ml. of sodium hydroxide of similar concentration to that in the pyrogallol solution, so that both treatments were free from carbon dioxide and provided conditions of similar relative humidity. In order to displace the air in the anaerobic series a rapid stream of nitrogen was blown through the desiccators for half an hour. The efficiency of this procedure for displacing the air was tested by analysing samples of gas from the desiccators using a Haldane apparatus. In two experiments in which leaf disks were treated in the prescribed manner, duplicate analyses were carried out on each sample withdrawn from each desiccator immediately after air displacement and again at the end of a 10 hr. experimental period. Samples were also taken from the gas cylinder directly to check the composition of the gas used. Since the capacity of the Haldane apparatus was 10 ml. and the smallest measurable volume ml., it will be seen from Table i that the percentages of oxygen or carbon dioxide in the samples were near the limits of accuracy of the apparatus. The mean percentage of oxygen inside the desiccators after air displacement was 0-03 %. It was concluded that blowing nitrogen through the desiccators for 30 min. produced sufficiently anaerobic conditions, considering that in addition the gas was to stand over alkaline pyrogallol during the experiment. In the first experiment there appeared to be more oxygen inside the desiccator at the end of the experimental period. The reason for this is difficult to see in view of the presence of the alkaline pyrogallol. In the second experiment no oxygen was detectable at the end of the experiment. Table I Volumes in ml. Volume of gas taken Volume of gas after CO2 absorption Volume of gas after Oj absorption Volume of COj Volume of O2 % CO2 Na direct from cylinder a b After air displacement (i) Gas from desiccators (2) After 10 hr. experiment (I) o-oii 0-15 (2) a and b were duplicate analyses, (i) and (2) were separate experiments of which the results given are means of two analyses each. EXPERIMENTS (i) Comparison of uptake of sucrose in air and nitrogen One sample of thirty-six disks was floated on 10% sucrose solution, and a second floated on water. Both were placed in a desiccator and the air replaced by nitrogen in the manner described above. A second pair of samples was floated similarly inside a desiccator containing air. The experiment was duplicated and was carried out in a dark room at a temperature of about 25" C. (no precise control). After floating for 10 hr. the disks were removed and their dry weights and water-contents measured. The results are set out in Table 2. The mean dry weight after floating on sucrose solution in air was 78-5 mg. compared with 58-5 mg. on water, indicating a sucrose

3 Uptake of sucrose and water by floating leaf disks 15 uptake of 20 mg. Under anaerobic conditions uptake was 5-5 mg. Thus anaerobic conditions reduced sucrose uptake by nearly 75 %. A number of experiments of similar pattern have given essentially similar results, the ratio of uptake in air to nitrogen being about 4 to I. Table 2. Weights of disks in mg. after floating for 10 hr. on water or 10% sucrose solution Final dry weight Mean Uptake of sucrose \ f Final water content \ f Mean water" content Water Air 2C -0 Sucrose soln a and b refer to duplicate samples. Water Nitrogen 5 5 Sucrose soln. With regard to the water-content of the samples, those disks which had been floated on water were presumably fully turgid and there was little difference between the air and nitrogen treatments (314 and 309 mg. respectively). The water-content of those disks which had been floated on sucrose solution in air was also about the same (313 mg.) whereas the water-content of those in nitrogen had declined to 291 mg. This last figure presumably represented osmotic equilibrium with the external solution (10% sucrose) whilst the uptake of sucrose in air had permitted the water-content of the disks to attain equality with that of fully turgid disks, i.e. full recovery had taken place in 10 hr. It might be supposed that the action of nitrogen in inhibiting sucrose uptake was merely an injurious one irreversibly damaging the protoplasm. That this was not so was demonstrated in the following experiment, in which the uptake was followed for a time in anaerobic conditions with subsequent transference to aerobic conditions, and in aerobic conditions with subsequent transference to anaerobic conditions. Ten 40-disk samples were collected. Four were floated on 10 % sucrose solution in air, four in nitrogen and two were used directly for obtaining an initial value of dry weight and water-content. After 3I hr. one sample was withdrawn from the air and nitrogen series and dry weights and water-contents measured. Second samples were also withdrawn after 7 hr. The remaining two samples in the nitrogen series were then transferred to air and one withdrawn 3I hr., the other 7 hr., after transference. The remaining two samples in the air series were treated similarly except that they were transferred to nitrogen. The results are plotted in Fig. i. During the first 7 hr. there was a rapid uptake of sucrose in air and a correspondingly small uptake in nitrogen. On transference to nitrogen the disks which had been floated in air showed little further uptake of sucrose. Those disks which had been in nitrogen, on transference to air, absorbed sugar rapidly, the rate being approximately equal to the initial uptake of the air series, and the final dry weights in the two series being very nearly equal. It is concluded from this that the inhibition of sucrose uptake by nitrogen was reversible and could not be ascribed to an injurious effect on the cells

4 i6 P. E. WEATHERLEY 40 b^-^ Time (hr.) Fig. I. Increase in dry weight of leaf-disk samples floated on io% sucrose solution under aerobic and anaerobic conditions with reversal of conditions after 7 hr. (2) The course of uptake of sucrose and changes in water-content of floating leaf disks {a) In air A number of samples were floated on sucrose solution and pairs of samples withdrawn from time to time for dry weight and water-content measurement. Ten samples, each of 40 disks, were first floated on water for 24 hr. to bring them to full turgidity. Two samples were then chosen at random for initial dry weight and water-content measurement. The remainder were floated on Petri dishes inside desiccators containing sodium hydroxide solution as in the air series described above. The experiment was carried out in continuous artificial light. A random pair of samples was withdrawn for dry weight and watercontent measurement after 2, 5, io and 22 hr. Results are plotted in Fig. 2. It will be seen that there was a steady rise in dry weight as a result of sucrose uptake, the rate falling off a little with time. The water-content of the turgid disks fell on being placed on the sucrose solution and reached a minimum sometime between 2 and 5 hr., after which the water-content began to rise, complete recovery to the turgid water-content being achieved after about 7 hr. Thereafter the water-content of the tissues continued to rise steadily, reaching almost 17% in excess of the turgid value after 22 hr. These results may be interpreted as an active transfer of sucrose into the cells, the movement of water following passively. Initially exosmosis of water took place, the diffusion potential deficit (D.P.D.) of the 10% sucrose solution being greater than that of the tissues (zero at full turgidity).

5 uptake of sucrose and water by floating leaf disks 17 At the minimum water-content the D.P.D. of the tissues had attained equality with that of the external solution, in part by the cells having contracted in volume, in part by the absorption of solute. Thereafter continued absorption of sucrose raised the D.P.D. of the cells above that of the external solution and water began to enter the cells again. At first the water uptake was presumably slow, but gradually became more rapid as the difference in diffusion potential between the tissue and the external solution increased, until finally a steady state was achieved in which the uptake of water balanced the uptake of sugar. - _ _- - Dry weight - Water content X ' Jf^ X ^^^^^- 350 ^^.^ v 90 - a 80 _ _ \y \ ;- \\ 1 ^ - ^--^ 1 I 1 X ' Hours Turgid water content _ C o u I- _ <u j Fig. 2. Course of dry-weight increase and change in water-content of initially turgid leaf disks floated on 10% sucrose solution. Each cross is the mean of two values shown as points on either side of it. This was the position during the period from 10 to 22 hr. when there was a steady uptake of sucrose accompanied by a steady absorption of water, in this experiment 2-5 mg. of water accompanying every mg. of sucrose entering the cells. This means that each mg. of sucrose entering the cells lowered the diffusion potential of the vacuolar water by the same amount as it was raised by the entry of 2-5 mg. of water in increasing the hydrostatic pressure in the cells and diluting their vacuolar contents. This ratio of water to sugar entering the cells would not be constant even within a single experiment. It would be expected to decline as the volume of the cells increased. That it in fact did so may be seen in Fig. 3, in which the changes in water-content and dry weight of samples fioated on 10 % sucrose solution and water are plotted over 70 hr. Between 21 and 45 hr. water uptake per unit uptake of sucrose was 2-98, whilst between 2 New Phyt. 54, i

6 i8 P. E. WEATHERLEY 45 and 70 hr. when the cells were more distended and more strongly resisted any further increase in volume, the ratio had fallen to o-6i. Nevertheless, during a period of i or 2 hr. between the time limits of say 1(^40 hr. the ratio did not appear to change appreciably, and during such a time interval there was presumably a steadily maintained difference of diffusion potential between the tissues and the medium. On sucrose solution On water 0-0 I I I I Fig. 3. Change in dry weight and water-content of initially turgid leaf disk floated on water and 10% sucrose solution. In order to ascertain this difference, the D.P.D. of the disks during sugar uptake was measured in the following way. Twelve samples were floated on 10% sucrose solution for 21 hr. after which they were weighed and divided into pairs at random. The watercontent of one pair was measured directly, a second pair was floated on 10% sucrose solution as before, and the remaining four pairs were floated on 3, 6, 9 and 12 % mannitol* solutions. These last four pairs were weighed after i hr. on the mannitol solutions, and after 2 further hours all samples were weighed and water-contents measured. The results are shown in Table 3. It will be seen that there was a fairly large increase of dry weight * Mannitol penetrates the cells to a less extent than sucrose.

7 Uptake of sucrose and water by floating leaf disks 19 on the mannitol indicating its penetration into the cells. It was desirable, therefore, to use changes in water-content rather than fresh weight for computing the D.P.D. of the tissue, except for the i hr. values where the dry-weight increments were presumably less and the fresh-weight changes corresponded more closely to changes in water-content. Table 3. Changes in fresh weight and water-content with dry weights of disks transferred to sucrose and mannitol solutions after floating for 21 hr. on 10% sucrose solution Data in mg. Change in fresh weight after i hr 1 a \b Change in water content after 3 hr. \a \b Dry weight (mean of 2 values) Initial value 73-2 Sucrose 10% % I S a and b refer to duplicate sets of samples. 6% I Mannitol 9% -t-i Z % After 3 hr. After 1 hr I. _L _L _L Percentage concentration of mannitol solution Fig. 4. Graph showing change in water-content (3 hr. curve) and fresh weight (i hr. curve) of leaf disks plotted against concentration of plasmolyticum. Each point is the mean of the two values shown on either side of it. Changes in water-content are plotted against concentration of mannitol in Fig. 4. It is evident that the curve for the i hr. values is of different form from the 3 hr. curve, and gives a higher value for the D.P.D. of the tissues. In the former case it is equivalent to a 10-5 % mannitol solution or 11-7 atm., in the latter to 8-25 % mannitol or 10-2 atm. Any effect of the penetration of mannitol would have altered the magnitude of these values in

8 2O P. E. WEATHERLEY the opposite sense: the value after 3 hr. would have been greater than the value after i hr. This decline in the D.P.D. during the 3 hr. following floating on sucrose solution might have been due to sugar transformations inside the cells resulting in the formation of less osmotically active substances such as starch. At all events the D.P.D. of the disks would seem to have been at least 3-6 atm. greater than that of the 10% sucrose solution (6-6 atm.) on which they had been floating for 21 hr. It is also evident that as the volume of the cells increased, and with it the reaction of the cell walls, the maintenance of a D.P.D. greater than that of the external solution must have required a steady increase in osmotic potential of the vacuolar sap Hours Fig. 5. Change in dry weight and water-content of leaf disks with an initial water deficit floated on water and 10% sucrose solution. A second experiment on the course of uptake of sucrose and water gave similar results to that described above. In this the rate of uptake of sucrose was less than in the first whilst the other differences were the expected results of this. Thus the minimum volume attained was lower, and the time taken to regain the turgid water-content longer, than in the first experiment. In a third experiment the course of changes in dry weight and water-content was followed in leaf disks which were not fully turgid to start with. In this experiment parallel samples were floated on water in addition to those on 10% sucrose solution. Results are plotted in Fig. 5. The changes in dry weight were as in the previous experiments. The dry weight changed little on water, whereas on sucrose solution there was an increase in dry weight of almost 80 % in 22 hr. The water-content of disks floated on water increased rapidly during the first 2 hr. and thereafter increased very slowly, the

9 uptake of sucrose and water by floating leaf disks 21 disks being turgid. The water-content of the sucrose series fell very slightly at first but soon began to increase, indicating that the D.P.D. of the tissue was initially not very different from that of the 10% sucrose solution. The water-content attained equality with the turgid disks on water in about 8 hr. and continued to increase steadily, so that after 22 hr. the water-content of the disks on sucrose solution was 15 % higher than that of disks floated on water. {b) In nitrogen In these experiments the course of changes in dry weight and water-content was followed in turgid disks floated on 10% sucrose solution in an atmosphere of nitrogen with an air series for comparison. These experiments were carried out in the dark since, as will be shown in a subsequent publication, light has a profound effect on the behaviour of the tissues in nitrogen. Ten samples of 40 disks each were collected. Four samples were floated on sucrose solution in four separate nitrogen-filled desiccators. A second group of four samples was similarly floated in air-filled desiccators. One sample was treated immediately for dryweight and water-content measurement, and one sample was floated on water in air. One sample was withdrawn from the nitrogen group and one from the air group after 2, 5, 10 and 23 hr. floating. At the 23 hr. withdrawal the water-floated sample was also withdrawn. It will be seen that the experiment was not replicated owing to the difliculty of taking more samples from one set of leaves, but the magnitude of the errors was known from the many replicated experiments with disks sampled in the same way. Results are plotted in Fig. 6. In air there was a steady increase in dry weight. At the end of 23 hr. this amounted to a 45 % increase in dry weight or an uptake of 36 mg. of sucrose. In nitrogen there appeared to be a slight increase during the first 10 hr. and then a decline so that the dry weight of the final sample was only 2 mg. above the initial one. Floating on water for the period of the experiment led to a slight decline in dry weight. The water-content in the air series followed the pattern already described, complete recovery to the turgid water-content was attained in about 9 hr., and by the end of the experiment there was a considerable excess water-content over the sample floated on water. In nitrogen the behaviour of the disks was that expected of tissues immersed in a hypertonic solution of a non-penetrating solute. Exosmosis occurred and the watercontent of the tissues declined and attained an equilibrium after some 10 hr. as a watercontent of 250 mg. compared with a turgid value of 285 mg. This represented a decline in volume of about 12% for a change in diffusion potential of 6-6 atm. (3) Computation of permeability constants for water and sucrose Since reliable measurements of permeability constants of plant cells to water and solutes are diflicult to make, it is worth examining the data presented here to see whether they permit the evaluation of such constants. Jacobs (1933) has devised a method for measuring the permeability constants of sea-urchin eggs to water and a given solute. The eggs are immersed in a hypertonic solution containing the penetrating solute. If the initial volume of a given cell is known, together with the minimum volume attained prior to recovery, and the time taken to reach this minimum volume, the permeabihty constants of the cell to the solute and water can be calculated. Since these data are known for the leaf disks under investigation here, it might be thought that this method could be used.

10 Fig. 6. Change in dry weight and water-content of disks floated on water and io% sucrose solution in air and nitrogen.

11 uptake of sucrose and water by floating leaf disks 23 It cannot, however, be applied to plant cells in a turgid state owing to the part played by the cell wall in contributing to the D.P.D. in the vacuole. A more direct approach is possible. When the disks have been floated on sucrose solution long enough to pass the minimum volume there is a steady absorption of water in response to a steady uptake of sucrose. Over a period of an hour or two there is little variation in this rate, and it seems reasonable to assume that there is a steady difference of diffusion potential between the medium and the vacuoles. If this difference is known, together with the rate of water absorption and surface area over which absorption is taking place, the value of the permeability constant for water may be calculated: r_ V ta{p^-p;)' where -^=permeability constant, F= volume of water {fi^) entering the tissue in t min., A = area of absorbing surface {j.^), Pj, = D.P.D. of tissue, Pj = D.P.D. of external sucrose solution. All these data were obtained in the experiment described in 2 (a) above. In this experiment the D.P.D. of the tissue during steady absorption of water was 10-2 atm., that of the external solution 6-6 atm., giving a difference of 3-6 atm. During the 3 hr. period of the experiment 10-7 mg. or 1-07 x io^v^ of water were absorbed. It only remained to measure the surface of absorption. It has been shown (Weatherley, 1947, 1954) that absorption is through the cut edges of leaf disks. The mean diameter of the disks was measured, using a travelling microscope, and their mean thickness by cutting sections and measuring with a microscope with micrometer eye-piece. The mean diameter was found to be 8-09 mm. and the mean thickness ^^- Thus the area of the cut edge was 77 x 8-09 x mm.^ or 6-22 mm.^. For a whole sample of twenty-five disks this becomes: 25 X 6-22 mm.^ or 1-56: Thus i'o7 X io^" 3 X 60 X 1-56 X 3-6 X = 0- The corresponding permeability constant of the tissue to sucrose cannot be computed accurately since the internal concentration of sucrose is not known. Nevertheless, a value can be estimated if it is assumed that the internal concentration was being maintained at zero. The rate of uptake of sucrose was approximately i mghr. or 1342 x 1000 g. mol.hr., the concentration gradient g. mol.l. The area of absorption was as in the previous calculation. Thus ^ ^ 1x3-42 ^^ ' 342x1000x60x1-56x10* = 1-07 X 10-^^ g. mol.x^min.molar concentration difference. This value might well be an underestimate, for the internal concentration was likely to have been above zero and the concentration gradient less than Also the uptake of I mg.hr.sample was somewhat below average, rates of double this value having been observed. Against this the area of uptake might have been greater, for example the solution might have been taken in through the cut ends of the veins and distributed by them through the tissues. This does not seem likely however, for reference to Fig. 2

12 24 p. E. WEATHERLEY shows that a steady rate of sucrose uptake took place whether water was passing in or out of the disks. Had sucrose solution been taken in through the veins, uptake would have been greater when water was being absorbed by the disks. Also the xylem elements of cut disks might well be air-filled and therefore non-conducting. It seems reasonable to conclude that the value 1-07 x io-^^ is at least not an overestimate for the permeability constant of the disks to sucrose. (4) The effect of sucrose uptake on the measurement of the diffusion potential deficit of tissues Since sucrose solutions are often used as plasmolytica in the measurement of osmotic potential or diffusion potential deficit of plant tissues, it is of interest to see what error is introduced by their use on a tissue as permeable to sucrose as that of Atropa leaves. Fresh weight Water content Osmotic potential in atmospheres Fig. 7. Change in water-content and fresh weight of disks after submergence for 3 hr. in four graded sucrose solutions. The disks were previously equilibrized on mannitol solution. The change in weight after 3 hr. submergence in this same mannitol solution is also shown To investigate this point the D.P.D. of a number of leaf-disk samples was brought to a known value (3-7 atm.) by floating them on 3 % mannitol solution. Then the D.P.D. was found experimentally by measuring the change in fresh weight or water-content after submergence for 3 hr. in a graded series of sucrose solutions. Twelve 25-disk samples were used. After equilibration on the 3 % mannitol solution the initial water-content of one pair of samples was found by oven drying. Of the remaining five pairs, four were submerged in 5, 7, 10 and 14% sucrose solutions, whilst one pair was submerged in 3% mannitol solution again. The submerged disks were brightly illuminated to prevent injection of the intercellular spaces. After 3 hr. the samples were removed and fresh weights, dry weights and water contents found.

13 uptake of sucrose and water by floating leaf disks 25 In Fig. 7 changes in fresh weight and water-content are plotted against the osmotic potential of the plasmolyticum. It will be seen that the samples in 3 % mannitol solution showed little mean change in water-content indicating that the D.P.D. of the tissue was near this value. If the changes in water-content are used to measure the D.P.D. the line of nearest fit gives a value by interpolation of 5-6 atm., a value in error of the true value by more than 50%. The changes in fresh weight give an irregular curve which is much wider of the true value. This was because all the samples had absorbed sucrose and increased appreciably in dry weight during the 3 hr. of submergence in sucrose. DISCUSSION On the basis of the foregoing results it seems to be a reasonable hypothesis that sugar is absorbed actively and water follows passively. That the uptake of sugar was active is based on three pieces of evidence. First, the value for the permeability constant (-Kguc. = i' 7 X 10-^^ g. mol.x^min.mol.l. concentration difference) even though it is likely to be an underestimate, is considerably higher than values published for other plant tissues. Davson & DanieUi (1943) give values for algal cells ranging between io~^' and io~2". Cells of higher plants seem to be in the same range (Hofmeister, 1941). This is not in line with the uptake by Atropa leaves being a process of diffusion through lipoid membranes, but points to an active process. Secondly, the uptake is reversibly inhibited under anaerobic conditions. This is consistent with the hypothesis of the uptake being an energy-using process requiring aerobic respiration. Strength is given to this view by the fact that the sugar uptake is almost entirely inhibited by mercury vapour (Pennel & Weatherley, 1954) emphasizing the probable importance of enzymic SH groups to the uptake. Of course this is little more than supporting evidence, for a change in rate of penetration of a substance can be brought about merely by a change in permeability of the membrane. It is known that the permeability of plasma membranes is very sensitive to external changes such as in the partial pressure of oxygen. In the case of these Atropa leaf disks a considerable cut-down in sugar uptake under anaerobic conditions might be due to fall in permeability of the tissue, the uptake being a purely diffusional one. It is interesting in this connexion that the rate of diffusion of sucrose through a 5 m^t. layer ofwaer is 4-5 x io-^mol.min.^a^unit molar concentration difference (Davson & DanieUi, 1943) or about 10^^ times faster than through a lipoid membrane. There is thus scope for a small change in membrane structure enormously altering its permeability. Conclusive evidence for an active transfer of a solute can only come by a demonstration of movement of the solute across the membrane in a way contrary to expectation on a purely diffusional hypothesis, e.g. against a concentration gradient. In these experiments it has not been possible to demonstrate that the sucrose was transferred into the cells against a concentration gradient, since the internal concentration of sucrose was not known. But absence of leakage when the tissue is in contact with distilled water would be evidence of a similar kind. If the measured uptake of sugar were due to diffusion, on water a leakage of sugar into the water at a comparable rate would be expected. No specific experiments on leakage have been carried out, but no rapid decline in dry weight has been observed in disks collected from the plant and floated directly on water, nor when such water was evaporated to dryness was it found to contain any weighable solids (Weatherley, 1954)- This points to an absence of leakage and a retention of sugars

14 26 p. E. WEATHERLEY inside the cells against a concentration gradient. It might be expected that in an atmosphere of nitrogen, with an inhibition of active mechanisms, leakage would become pronounced. Reference to Fig. i shows that disks which had been floating on sugar solution in air did not show a marked decline in weight on transference to nitrogen, i.e. there was little evidence for leakage. This apparent retention of sugar, even under anaerobic conditions, could be explained if sucrose were converted inside the cell into substances unable to penetrate the membrane, e.g. starch. In this way the internal concentration of sugar would remain low and absorption by diffusion continue steadily. Yet all of such accumulating substances could not have been insoluble, since a marked rise in the osmotic potential of the sap occurred during sucrose uptake indeed a rough calculation indicates that the rise in osmotic potential of the sap could not be accounted for even in terms of all the sucrose absorbed. This implies a breakdown of the sucrose, or part of it, into smaller molecules, e.g. hexose, inside the cell. But if the solute exchanges were purely diffusional, these smaller molecules would be expected to leak out of the cells even more rapidly than sucrose. Perhaps the absence of leakage under anaerobic conditions points to a process of active transfer into the cells needing aerobic conditions, but a process of retention inside the cells not dependent on aerobic processes. The third line of evidence for the uptake of sucrose being an active process is, then, the absence of leakage of sugar when the tissue was floated on water. Yet the question of leakage from tissues which have accumulated carbohydrate clearly needs further investigation, together with the nature of the substances accumulating inside the cells. In sum, the three lines of evidence point to an active uptake of sucrose by the leaf disks, but as it stands this deduction is far from conclusive. No claim is made for the accuracy of the water permeability constant of the cells, though it would appear to be a maximal value and therein lies its interest, for it is low compared with most published values. The two major sources of error lay in the measurement of the surface of absorption and the potential gradient. It is likely that the circumference of a disk + its thickness, underestimates the absorptive surface, for some marginal injection of the intercellular spaces occurs which might increase the effective area of absorption. It has been shown above that the D.P.D. of the tissue was probably underestimated since there were indications of a decline in this value during the process of measurement. Thus the steady state gradient of potential along which water was moving into the disks was probably greater than that actually estimated. The underestimation of both these factors means that the computed value of i^h^o is probably too high. This value of O-I jumin.atm. is in itself about half the figure for the permeability constants of plant cells found in the literature. This is consistent with the hypothesis that whilst uptake of sucrose was active, the water accompanied it by diffusion. This picture is analogous with that which has been worked out in recent years for the transfer of ions and water into root systems. Lundegardh (1946, 1950) envisages a centripetal active transfer of salts in roots, water moving osmotically in response to this, whilst Arisz, Helder & van Nie (1951) have provided convincing experimental evidence that root bleeding is due to the secretion of salts into the xylem elements accompanied by a purely osmotic water movement. The data presented in this paper suggest that another mechanism exists in plants in which the active part played by salts is replaced by sugar. It is tempting to push the analogy with Lundegardh's picture even'further. At high oxygen pressures accumulation of salts by cells predominates, whilst at low oxygen

15 uptake of sucrose and water by floating leaf disks 27 pressures passive leakage occurs. In this way he accounts for the migration of ions and water across the root cortex as a result of its centripetal gradient of oxygen pressure. In this paper it is shown how the accumulation of carbohydrate is similarly related to the presence or absence of oxygen. It seems likely that if a tissue were situated in an environment of high oxygen pressure it would accumulate sugar, whilst if in a situation of low oxygen pressure it would not. This could have significance in connexion with the transport of sugar in the sieve tubes. In the leaves there would be active accumulation of sugars building up a solution of high concentration in the sieve tubes which would be translocated by mass flow to storage organs, roots, etc., where the oxygen pressure might well be low and where passive exudation of sugar out of the sieve tubes would occur. It is unlikely that the error introduced by using sucrose as a plasmolyticum for measuring osmotic quantities would be as great for plant tissues in general as it is for Atropa leaves. Nevertheless, it has been shown (Weatherley, 1954) that leaf tissues from various species do absorb sucrose, and the errors that would be introduced in these cases could hardly be ignored. This serves to emphasize a point made by Lundegardh (1950) that a careful examination of a tissue for the possible existence of a power to accumulate a plasmolytical agent is needed before postulating any anomalous components of osmosis on the basis of the use of that plasmolytical agent. SUMMARY 1. The uptake of sucrose by floating leaf disks of Atropa belladonna, measured as increase in dry weight, was compared under aerobic and anaerobic conditions. In experiments of 10 hr. duration the uptake in air was about 4 times that in nitrogen. 2. The inhibition of uptake by nitrogen was reversible, the rate of uptake in air being unaflected by a previous period of 7 hr. in nitrogen. 3. Increase in dry weight and changes in water-content of leaf disks floating on 10 % sucrose solution was followed in both aerobic and anaerobic conditions. In air there was a steady increase in dry weight. The water-content, after falling during the first few hours, recovered and gained equality with fully turgid tissue after about 10 hr. Thereafter it continued to increase in response to the accumulation of solutes, to values far exceeding that of fully turgid tissue. During this period of accumulation of sucrose and water, the D.P.D. of the tissue was found to be a little over 10 atm. against a value of 6-6 atm. for the external sucrose solution, and 2-3 mg. of water was absorbed for each mg. of sucrose taken in. Under anaerobic conditions there was little uptake of sucrose, and the water-content of the disks behaved as if immersed in a hypertonic solution of a non-penetrating solute. 4. From the data obtained in these experiments permeability constants of the tissues for water and sucrose were computed. The value for water (o-ix^^a^atm.min.) is lower than that usually obtained for plant tissues, whereas that for sucrose (i x io-i^ g. mol.^^min.molar concentration diflerence) is much higher. 5. These results are explicable as an active uptake of sucrose accompanied by a passive absorption of water, although it is emphasized that the evidence for the active uptake of sucrose is far from conclusive. 6. It is suggested that this transfer of sugar followed passively by water is analogous with the salt transfer across the root cortex proposed by Lundegardh. The possibility of a similar transfer between points of high and low oxygen pressures is discussed.

16 28 p. E. WEATHERLEY 7. The errors introduced by using sucrose as a plasmolyticum for D.P.D. measurement of a tissue as permeable to sucrose as Atropa leaves are shown to be very considerable. REFERENCES ARISZ, N. H., HELDER, R. J. & NIE, R. VAN (1951). Analysis of the exudation process in tomato plants. jf. Exp. Bot. 2, 257. DAVSON, H. & DANIELLI, J. F. (1943). The Permeability of Natural Membranes. Cambridge University- Press. HoFMEiSTEH, L. (1941). Die Permeabilitat pflanzlichen Protoplasmas fiir Anelekrolyte. Tabiil. bid., Hague, 19(2), 263. JACOBS, M. H. (1933). The simultaneous measurement of cell permeability to water and to dissolved substances. J. Cell. Comp. Physiol. 2, 427. LuNDEGARDH, H. (1946). Transport of water and Salts through plant tissues. Nature, Lond., tgy, 575. LuNDEGARDH, H. (1950). The translocation of salts and water through wheat roots. Physiol. Plant. 3, 103. PENNEL, A. G. & WEATHERLEY, P. E. (1954). Natures, Lond., 173, WEATHERLEY, P. E. (1947). Note on the diurnal fluctuations in water content of floating leaf disks. New Phytol. 46, 276. WEATHERLEY, P. E. (1954). Preliminary investigations into the uptake of sugars by floating leaf disks. New Phytol. (in the Press).

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