THE INFLUENCE OF LIGHT,TEMPERATURE, AND OTHER CONDITIONS ON THE ABILITY OF NITELLA CELLS TO CONCENTRATE HALOGENS IN THE CELL SAP

1. By the use of a special analytical technique it has been possible to study the accumulation of halogens in the cell sap of Nitella. 2. From a dilute solution, Br may be accumulated in the sap in a concentration much greater than that of the external solution. The conductivity of the sap may be markedly increased by such accumulation. The process is a slow one so that a month or more may be required to approach equilibrium. 3. Cl may be lost from the cell as a result of the accumulation of Br and vice versa. Other reciprocal relations between Cl and Br are indicated. 4. At equilibrium practically as much Br accumulated in the sap with an external solution containing 1 milli-equivalent of Br as with one containing 5 milli-equivalents. 5. Light energy was indispensable to the accumulation of Br. The temperature coefficient was characteristic of a chemical process.     

ACCUMULATION OF BRILLIANT CRESYL BLUE IN THE SAP OF LIVING CELLS OF NITELLA IN THE PRESENCE OF NH3

When the living cells of Nitella are placed in a solution of brilliant cresyl blue containing NH₄Cl, the rate of accumulation of the dye in the sap is found to be lower than when the cells are placed in a solution of dye containing no NH₄Cl and this may occur without any increase in the pH value of the cell sap. This decrease is found to be primarily due to the presence of NH₃ in the sap and seems not to exist where NH₃ is present only in the external solution at the concentration used.     

THE PENETRATION OF BASIC DYE INTO NITELLA AND VALONIA IN THE PRESENCE OF CERTAIN ACIDS,BUFFER MIXTURES,AND SALTS

When living cells of Nitella are exposed to an acetate buffer solution until the pH value of the sap is decreased and subsequently placed in a solution of brilliant cresyl blue, the rate of penetration of dye into the vacuole is found to decrease in the majority of cases, and increase in other cases, as compared with the control cells which are transferred to the dye solution directly from tap water. This decrease in the rate is not due to the lowering of the pH value of the solution just outside the cell wall, as a result of diffusion of acetic acid from the cell when cells are removed from the buffer solution and placed in the dye solution, because the relative amount of decrease (as compared with the control) is the same whether the external solution is stirred or not. Such a decrease in the rate may be brought about without a change in the pH value of the sap if the cells are placed in the dye solution after exposure to a phosphate buffer solution in which the pH value of the sap remains normal. The rate of penetration of dye is then found to decrease. The extent of this decrease is the greater the lower the pH value of the solution. It is found that hydrochloric acid and boric acid have no effect while phosphoric acid has an inhibiting effect at pH 4.8 on stirring. Experiments with neutral salt solutions indicate that a direct effect on the cell (decreasing penetration) is due to monovalent base cations, while there is no such effect directly on the dye. It is assumed that the effect of the phosphate and acetate buffer solutions on the cell, decreasing the rate of penetration, is due (1) to the penetration of these acids into the protoplasm as undissociated molecules, which dissociate upon entrance and lower the pH value of the protoplasm or to their action on the surface of the protoplasm, (2) to the effect of base cations on the protoplasm (either at the surface or in the interior), and (3) possibly to the effect of certain anions. In this case the action of the buffer solution is not due to its hydrogen ions. In the case of living cells of Valonia under the same experimental conditions as Nitella it is found that the rate of penetration of dye decreases when the pH value of the sap increases in presence of NH₃, and also when the pH value of the sap is decreased in the presence of acetic acid. Such a decrease may be brought about even when the cells are previously exposed to sea water containing HCl, in which the pH value of the sap remains normal.     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

THE COMPOSITION OF THE CELL SAP OF THE PLANT IN RELATION TO THE ABSORPTION OF IONS

1. Chemical examination of the cell sap of Nitella showed that the concentrations of all the principal inorganic elements, K, SO₄, Ca, Mg, PO₄, Cl, and Na, were very much higher than in the water in which the plants were growing. 2. Conductivity measurements and other considerations lead to the conclusion that all or nearly all of the inorganic elements present in the cell sap exist in ionic state. 3. The insoluble or combined elements found in the cell wall or protoplasm included Ca, Mg, S, Si, Fe, and Al. No potassium was present in insoluble form. Calcium was predominant. 4. The hydrogen ion concentration of healthy cells was found to be approximately constant, at pH 5.2. This value was not changed even when the outside solution varied from pH 5.0 to 9.0. 5. The penetration of NO₃ ion into the cell sap from dilute solutions was definitely influenced by the hydrogen ion concentration of the solution. Penetration was much more rapid from a slightly acid solution than from an alkaline one. It is possible that the NO₃ forms a combination with some constituent of the cell wall or of the protoplasm. 6. The exosmosis of chlorine from Nitella cells was found to be a delicate test for injury or altered permeability. 7. Dilute solutions of ammonium salts caused the reaction of the cell sap to increase its pH value. This change was accompanied by injury and exosmosis of chlorine. 8. Apparently the penetration of ions into the cell may take place from a solution of low concentration into a solution of higher concentration. 9. Various comparisons with higher plants are drawn, with reference to buffer systems, solubility of potassium, removal of nitrate from solution, etc.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : IV. PENETRATION OF TRIMETHYL THIONIN INTO NITELLA AND VALONIA FROM METHYLENE BLUE.

Spectrophotometric measurements show that it is chiefly the trimethyl thionin that is present in the sap extracted from the vacuoles of uninjured cells of Nitella or Valonia which have been placed in methylene blue solution at a little above pH 9. Whether these measurements were made immediately or several hours later the same results were obtained. Methylene blue is detected in the sap (1) when the cells are injured or (2) when the contamination of the sap from the stained cell wall occurs at the time of extraction. The sap is found to be incapable of demethylating methylene blue dissolved in it even on standing for several hours. It is somewhat uncertain as to whether the trimethyl thionin penetrated as such from the external methylene blue solution which generally contains this dye as impurity (in too small concentration for detection by spectrophotometer but detectable by extraction with chloroform), or whether it has formed from methylene blue in the protoplasm. The evidences described in the text tend to favor the former explanation. Theory is discussed on basis of more rapid penetration of trimethyl thionin (in form of free base) than of methylene blue, or of trimethyl thionin in form of salt.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : V. WHY DOES AZURE B PENETRATE MORE READILY THAN METHYLENE BLUE OR CRYSTAL VIOLET?

Glass electrode measurements of the pH value of the sap of cells of Nitella show that azure B in the form of free base penetrates the vacuoles and raises the pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, but the dye salt dissolved in the sap does not alter the pH value of the sap. It is concluded that the dye penetrates the vacuoles chiefly in the form of free base and not as salt. The dye from methylene blue solution containing azure B free base as impurity penetrates and accumulates in the vacuole. This dye must be azure B in the form of free base, since it raises the pH value of the sap to about the same extent as the free base of azure B dissolved in the sap in vitro. The dye absorbed by the chloroform from methylene blue solution behaves like the dye penetrating the vacuole. These results confirm those of spectrophotometric analysis previously published. Crystal violet exists only in one form between pH 5 and pH 9.2, and does not alter the pH value of the sap at the concentrations used. It does not penetrate readily unless cells are injured. A theory of "multiple partition coefficients" is described which explains the mechanism of the behavior of living cells to these dyes. When the protoplasm is squeezed into the sap, the pH value of the mixture is higher than that of the pure sap. The behavior of such a mixture to the dye is very much like that of the sap except that with azure B and methylene blue the rise in the pH value of such a mixture is not so pronounced as with sap when the dye penetrates into the vacuoles. Spectrophotometric measurements show that the dye which penetrates from methylene blue solution has a primary absorption maximum at 653 to 655 mµ (i.e., is a mixture of azure B and methylene blue, with preponderance of azure B) whether we take the sap alone or the sap plus protoplasm. These results confirm those previously obtained with spectrophotometric measurements.     

SPECTROPHOTOMETRIC STUDIES OF PENETRATION : V. RESEMBLANCES BETWEEN THE LIVING CELL AND AN ARTIFICIAL SYSTEM IN ABSORBING METHYLENE BLUE AND TRIMETHYL THIONINE.

The rate of diffusion through the non-aqueous layer of the protoplasm depends largely on the partition coefficients mentioned above. Since these cannot be determined we have employed an artificial system in which chloroform is used in place of the non-aqueous layer of the protoplasm. The partition coefficients may be roughly determined by shaking up the aqueous solutions with chloroform and analyzing with the spectrophotometer (which is necessary with methylene blue because we are dealing with mixtures). This will show what dyes may be expected to pass through the protoplasm into the vacuole in case it behaves like the artificial system. From these results we may conclude that the artificial system and the living cell act almost alike toward methylene blue and azure B, which supports the notion of non-aqueous layers in the protoplasm. There is a close resemblance between Valonia and the artificial system in their behavior toward these dyes at pH 9.5. In the case of Nitella, on the other hand, with methylene blue solution at pH 9.2 the sap in the artificial system takes up relatively more azure B (absorption maximum at 650 mµ) than the vacuole of the living cell (655 mµ). But both take up azure B much more rapidly than methylene blue. A comparison cannot be made between the behavior of the artificial system and that of the living cell at pH 5.5 since in the latter case there arises a question of injury to cells before enough dye is collected in the sap for analysis.     

EXIT OF DYE FROM LIVING CELLS OF NITELLA AT DIFFERENT pH VALUES

Experiments on the exit of brilliant cresyl blue from the living cells of Nitella, in solutions of varying external pH values containing no dye, confirm the theory that the relation of the dye in the sap to that in the external solution depends on the fact that the dye exists in two forms, one of which (DB) can pass through the protoplasm while the other (DS) passes only slightly. DB increases (by transformation of DS to DB) with an increase in the pH value, and is soluble in substances like chloroform and benzene. DS increases with decrease in pH value and is insoluble (or nearly so) in chloroform and benzene. The rate of exit of the dye increases as the external pH value decreases. This may be explained on the ground that DB as it comes out of the cell is partly changed to DS, the amount transformed increasing as the pH value decreases. The rate of exit of the dye is increased when the pH value of the sap is increased by penetration of NH₃.     

THE KINETICS OF PENETRATION : XV. THE RESTRICTION OF THE CELLULOSE WALL

When Valonia cells are impaled on capillaries, it is in some ways equivalent to removing the comparatively inelastic cellulose wall. Under these conditions sap can migrate into a free space and it is found that on the average the rate of increase of volume of the sap is 15 times what it is in intact cells kept under comparable conditions. The rate of increase of volume is a little faster during the first few hours of the experiment, but it soon becomes approximately linear and remains so as long as the experiment is continued. The slightly faster rate at first may mean that the osmotic pressure of the sap is approaching that of the sea water (in the intact cell the sap osmotic pressure is always slightly above that of the sea water). This might result from a more rapid entrance of water than of electrolyte, as would be expected when the restriction of the cellulose wall was removed. During the linear part of the curve the osmotic concentration and the composition of the sap suffer no change, so that entrance of electrolyte must be 15 times as fast in the impaled cells as it is in the intact cells. The explanation which best accords with the facts is that in the intact cell the entrance of electrolyte tends to increase the osmotic pressure. As a consequence the protoplasm is partially dehydrated temporarily and it cannot take up more water until the cellulose wall grows so that it can enclose more volume. The dehydration of the protoplasm may have the effect of making the non-aqueous protoplasm less permeable to electrolytes by reducing the diffusion and partition coefficients on which the rate of entrance depends. In this way the cell is protected against great fluctuations in the osmotic concentration of the sap.     

Studies on the electrical properties of a single plant cell; internodal cell of Nitella flexilis

Impedance changes of single plant cells of Nitella flexilis were studied under different environmental conditions. With the analysis presented changes in resistance of the protoplasmic membrane and of cell sap can be studied independently and simultaneously. Under "transcellular osmosis," the resistance of the protoplasmic membrane and of the cell sap increase at the part of the cell where water enters, while they decrease where water goes out. Ethanol of low concentration (below 8 per cent) first decreases and later increases the resistance of the protoplasmic membrane. Concentrated ethanol (over 10 per cent), however, brings about a large decrease in resistance of the protoplasmic membrane. Its time course is not simple, but undulatory changes occur. When ethanol is applied to one part of the cell, the resistance of the protoplasmic membrane shows a different type of change, which may be attributed to the local osmotic effect of ethanol; injury generally occurs with comparatively low concentration. Methanol, ethanol, and propanol have almost the same effect upon the cell, while butanol is toxic at the same concentration. When the cell dies, the resistance of the protoplasmic membrane decreases greatly, while the resistance of the cell sap increases to a level (several hundred kilo ohms or more), expected when external solution and cell sap are freely mixed with each other.     

FURTHER EXPERIMENTS ON THE ABSORPTION OF IONS BY PLANTS,INCLUDING OBSERVATIONS ON THE EFFECT OF LIGHT

1. The conditions of illumination were found to exert a very significant influence on absorption of ions from dilute solution by Nitella. These conditions were also found to influence the penetration of Br and NO₃ into the cell sap. 2. It is concluded that absorption of ions by plants from dilute solutions involves energy exchanges, with light as the ultimate source of the energy. It is suggested that the absorption is intimately related to growth and metabolism. 3. One ion may affect the removal from solution or penetration into the cell sap of another ion present in the same solution, even in solutions of extremely low concentration. It is probable that all three types of relations may exist—anion to anion, cation to cation, and anion to cation. 4. The sulfate and phosphate ions exerted far less influence on the absorption of nitrate than did chlorine and bromine ions. It is suggested as a possibility that sulfate does not penetrate readily to those surfaces at which chlorine, bromine, nitrate, and other ions may become effective.     

DISSIMILARITY OF INNER AND OUTER PROTOPLASMIC SURFACES IN VALONIA. II

In measurements of P.D. across the protoplasm in single cells, the presence of parallel circuits along the cell wall may cause serious difficulty. This is particularly the case with marine algae, such as Valonia, where the cell wall is imbibed with a highly conducting solution (sea water), and hence has low electrical resistance. In potential measurements on such material, it is undesirable to use methods in which the surface of the cell is brought in contact with more than one solution at a time. The effect of a second solution wetting a part of the cell surface is discussed, and demonstrated by experiment. From further measurements with improved technique, we find that the value previously reported for the P.D. of the chain Valonia sap | Valonia protoplasm | Valonia sap is too low, and also that the P.D. undergoes characteristic changes during experiments lasting several hours. The maximum P.D. observed is usually between 25 and 35 mv., but occasionally higher values (up to 82 mv.) are found. The appearance of the cells several days after the experiment, and the P.D.''s which they give with sea water, indicate that no permanent injury has been received as a result of exposure to artificial sap. If such cells are used in a second measurement with artificial sap, however, the form of the P.D.-time curve indicates that the cells have undergone an alteration which persists for a long time. On the basis of the theory of protoplasmic layers, an attempt has been made to explain the observed changes in P.D. with time, assuming that these changes are due to penetration of KCl into the main body of the protoplasm.     

A GLASS ELECTRODE APPARATUS FOR MEASURING THE pH VALUES OF VERY SMALL VOLUMES OF SOLUTION

A glass electrode apparatus is described with which pH measurements can be made with as small volumes as 2 drops (about 0.14 cc.) of solution. Using this apparatus the change of pH of the vacuolar sap of Nitella, due to the penetration of brilliant cresyl blue, has been readily followed. The sap and the dye have been found to poison the usual type of hydrogen electrode.     

THE EFFECT OF ACETATE BUFFER MIXTURES,ACETIC ACID,AND SODIUM ACETATE,ON THE PROTOPLASM,AS INFLUENCING THE RATE OF PENETRATION OF CRESYL BLUE INTO THE VACUOLE OF NITELLA

When living cells of Nitella are exposed to a solution of sodium acetate and are then placed in a solution of brilliant cresyl blue made up with a borate buffer mixture at pH 7.85, a decrease in the rate of penetration of dye is found, without any change in the pH value of the sap. It is assumed that this inhibiting effect is caused by the action of sodium on the protoplasm. This effect is not manifest if the dye solution is made up with phosphate buffer mixture at pH 7.85. It is assumed that this is due to the presence of a greater concentration of base cations in the phosphate buffer mixture. In the case of cells previously exposed to solutions of acetic acid the rate of penetration of dye decreases with the lowering of the pH value of the sap. This inhibiting effect is assumed to be due chiefly to the action of acetic acid on the protoplasm, provided the pH value of the external acetic acid is not so low as to involve an inhibiting effect on the protoplasm by hydrogen ions as well. It is assumed that the acetic acid either has a specific effect on the protoplasm or enters as undissociated molecules and by subsequent dissociation lowers the pH value of the protoplasm. With acetate buffer mixture the inhibiting effect is due to the action of sodium and acetic acid on the protoplasm. The inhibiting effect of acetic acid and acetate buffer mixture is manifested whether the dye solution is made up with borate or phosphate buffer mixture at pH 7.85. It is assumed that acetic acid in the vacuole serves as a reservoir so that during the experiment the inhibiting effect still persists.     

THE KINETICS OF PENETRATION : VIII. TEMPORARY ACCUMULATION

A model is described which throws light on the mechanism of accumulation. In the model used an external aqueous phase A is separated by a non-aqueous phase B (representing the protoplasm) from the artificial sap in C. A contains KOH and C contains HCl: they tend to mix by passing through the non-aqueous layer but much more KOH moves so that most of the KCl is formed in C, where the concentration of potassium becomes much greater than in A. This accumulation is only temporary for as the system approaches equilibrium the composition of A approaches identity with that of C, since all the substances present can pass through the non-aqueous layer. Such an approach to equilibrium may be compared to the death of the cell as the result of which accumulation disappears. During the earlier stages of the experiment potassium tends to go in as KOH and at the same time to go out as KCl. These opposing tendencies do not balance until the concentration of potassium inside becomes much greater than outside (hence potassium accumulates). The reason is that KCl, although its driving force be great, moves very slowly in B because its partition coefficient is low and in consequence its concentration gradient in B is small. This illustrates the importance of partition coefficients for penetration in models and in living cells. It also indicates that accumulation depends on the fact that permeability is greater for the ingoing compound of the accumulating substance than for the outgoing compound. Other things being equal, accumulation is increased by maintaining a low pH in C. Hence we may infer that anything which checks the production of acid in the living cell may be expected to check accumulation and growth. This model recalls the situation in Valonia and in most living cells where potassium accumulates as KCl, perhaps because it enters as KOH and forms KA in the sap (where A is an organic anion). In some plants potassium accumulates as KA but when HCl exists in the external solution it will tend to enter and displace the weaker acid HA (if this be carbonic it can readily escape): hence potassium may accumulate to a greater or less extent as KCl. Injury of the cell may produce a twofold effect, (1) increase of permeability, (2) lessened accumulation. The total amount of electrolyte taken up in a given time will be influenced by these factors and may be greater than normal in the injured cell or less, depending somewhat on the length of the interval of time chosen.     

THE KINETICS OF PENETRATION : XIX. ENTRANCE OF ELECTROLYTES AND OF WATER INTO IMPALED HALICYSTIS

When cells of Halicystis are impaled on a capillary so that space is provided into which the sap can migrate, the rate of entrance of water and of electrolyte is increased about 10-fold. In impaled Valonia cells the rate is increased about 15-fold. After a relatively rapid non-linear rate of increase of sap volume immediately after impalement (which may possibly represent the partial dissipation of the difference of the osmotic energy between intact and impaled cells) the volume increases at a linear rate, apparently indefinitely. Since the halide concentration of the sap at the end of the experiment is (within the limits of natural variation) the same as in the intact cell, we conclude that electrolyte also enters the sap about 10 times as fast as in the intact cell. As in the case of Valonia we conclude that there is a mechanism whereby in the intact cell the osmotic concentration of the sap is prevented from greatly exceeding that of the sea water. This may be associated with the state of hydration of the non-aqueous protoplasmic surfaces.     

STUDIES ON PENETRATION OF DYES WITH GLASS ELECTRODE : IV. PENETRATION OF BRILLIANT CRESYL BLUE INTO NITELLA FLEXILIS

Glass electrode measurements of the pH value of the sap of Nitella show that cresyl blue in form of free base penetrates the vacuoles and raises pH value of the sap to about the same degree as the free base of the dye added to the sap in vitro, while the dye salt dissolved in the sap does not alter its pH value. It is proved conclusively that the increase in the pH value of the sap is due only to the presence of the dye and not to some other alkaline substance. Spectrophotometric measurements show that the dye which penetrates the vacuole is chiefly cresyl blue. When the protoplasm is squeezed into the sap, the pH value of the sap is higher than that of the pure sap. Such a mixture behaves very much like the sap in respect to the dye.     

ON THE IMPORTANCE OF MAINTAINING CERTAIN DIFFERENCES BETWEEN CELL SAP AND EXTERNAL MEDIUM

A striking difference exists between the internal and external solution in the case of Valonia macrophysa. If this difference is abolished by placing cells in their own sap most of them quickly die. There is some ground for believing that the maintenance of differences between the sap and the external medium is of importance for vital processes. The sap of Valonia macrophysa is not a balanced solution in the ordinary sense and the question may be raised whether in general the interior of the cell requires a balanced solution in order to maintain life: or it may be that we must distinguish between internal and external balanced solutions.     

THE KINETICS OF PENETRATION : XIV. THE PENETRATION OF IODIDE INTO VALONIA

When 0.1 M NaI is added to the sea water surrounding Valonia iodide appears in the sap, presumably entering as NaI, KI, and HI. As the rate of entrance is not affected by changes in the external pH we conclude that the rate of entrance of HI is negligible in comparison with that of NaI, whose concentration is about 10⁷ times that of HI (the entrance of KI may be neglected for reasons stated). This is in marked contrast with the behavior of sulfide which enters chiefly as H₂S. It would seem that permeability to H₂S is enormously greater than to Na₂S. Similar considerations apply to CO₂. In this respect the situation differs greatly from that found with iodide. NaI enters because its activity is greater outside than inside so that no energy need be supplied by the cell. The rate of entrance (i.e. the amount of iodide entering the sap in a given time) is proportional to the external concentration of iodide, or to the external product [N⁺]_o [I^-l_o, after a certain external concentration of iodide has been reached. At lower concentrations the rate is relatively rapid. The reasons for this are discussed. The rate of passage of NaI through protoplasm is about a million times slower than through water. As the protoplasm is mostly water we may suppose that the delay is due chiefly to the non-aqueous protoplasmic surface layers. It would seem that these must be more than one molecule thick to bring this about. There is no great difference between the rate of entrance in the dark and in the light.