On the Influence of some Environmental Factors on the Osmotic Behaviour of Isolated Protoplasts of Allium cepa

1. Levitt, Scarth and Gibbs (1936) described a method for recording the osmotic behaviour of gymnoplasts, i.e. of protoplasts that, after plasmolysis, have been taken out of the cell wall; the record was obtained by photographing the gymnoplasts by means of a cinematographic camera attached to a microscope. This method was adopted by us, but instead of a single gymnoplast we photographed several at the same time. 2. The gymnoplasts we used, were isolated from the bulb scales of Allium cepa after plasmolysis in a solution containing 0.66 mol dextrose and varying concentrations of KC1 and CaCla. 3. We studied the speed with which water passes into the gymnoplasts when the latter are partially deplasmolysed, and estimated the volume to which they expand on account of the partial deplasmolysis. The latter was obtained by diluting the plasmolyte either with pure water or with water in which KG1 and CaCl2 were dissolved in the same concentration as in the plasmolyte. We also studied the volume changes of gymnoplasts that were more than once plasmolysed and deplasmolysed. 4. It appeared that the degree of permeability, i.e. the speed with which water passes into the gymnoplasts, can be expressed clearly by means of the half-time constant, h.v.c. (cf. Ch. II, § 2). 5. As it is essential that the gymnoplasts we use for our experiments, are perfectly spherical, they were always controlled as to their shape. They appeared to deviate from the spherical shape only when there were no K- and Ca-ions in the medium (cf. Ch. II, § 1). 6. At a constant temperature, the volume of the gymnoplasts does not change, even when they remain for a considerable time in the plasmolyte. Therefore it is to be assumed that neither dextrose nor salts pass from the latter into the gymnoplasts. Only when the gymnoplasts, because of an unsuitable composition of the medium, are damaged, endosmosis or intrability of these solutes may take place. 7. If the gymnoplast behaved as an ideal osmotic system, it would obey van ’t Hoff’s law. In our experiments, however, small deviations were found, the change in volume remaining, as a rule, somewhat below the expected value (Ch. Ill, § 4). The cause of these deviations was discussed (Ch. IV, § 2 A). 8. The influence of temperature as well as that of the ratio between the concentration of the K-ions and that of the Ca-ions, on the speed with which water passes into the gymnoplast and on the magnitude of the volume the latter ultimately reaches, were experimentally investigated (Ch. Ill, §§ 2, 4 and 6). 9. Temperature proved to have a marked effect on the magnitude of the ultimate volume. A temperature of 2° C proved to be the only one at which the observed value agrees with the calculated one. At higher temperatures it always remains below the expected value, and at 20° C the difference between the observed value and the calculated one reached its highest value (Ch. IV, § 2 B). It was argued that these differences may be due to the influence temperature exercises, by the intermediary of the ATP system, on the sol-gel equilibrium in the protoplasm (Ch. IV, § 2 B). 10. The K- and Ca-ions in the medium too exercised a measurable influence on the ultimate volume. The observed value was always smaller than the expected one, and the largest deficit was found when the concentrations of the K-and Ca-ions were equal (0.05 n). It was argued that it might be possible to explain this influence by assuming that in the plasma-membrane a charge mosaic is present, which would change under the influence of the K- and Ca-ions. The membrane potential would be lowest when the concentrations of the K- and Ca-ions are equal. Because of the membrane potentials that would arise when the concentrations of these ions are unequal, electro-endosmotic forces would come into action. The latter would be responsible for an increase of the amount of water that is absorbed, and this electro-endosmosis would, of course, increase with the membrane potential. 11. Within the range that was tested the ratio between the concentrations of the K- and Ca-ions was found to be of far greater importance than their absolute value. 12. The speed with which water passes into the gymnoplast, was found to be strongly dependent upon temperature. Up to the highest temperatures we could test, it was found to increase (Ch. Ill, § 2). The Q_10 is considerably higher than that of an ideal osmotic process. Factors that might be responsible for this higher value, were discussed (Ch. IV, § 3). 13. K- and Ca-ions also influence the speed with which water passes into the gymnoplast. When they are present at the same time, they show a distinct antagonism, similar to that which appears in their effect on phosphate colloids. The speed with which water passes into the gymnoplast, reaches its lowest value when the concentrations of the K- and Ca-ions are equal (0.05 n). The effect of an increase in the concentration of the K-ions accompanied by an equivalent decrease of the concentration of the Ca-ions, appears to be greater than that of a corresponding increase in the concentration of the Ca-ions accompanied by a similar decrease in the concentration of the K-ions. These facts form a strong argument in favour of the view that phosphate colloids (lecithin) play an important part in the composition of the plasma-membrane (Bungenberg de Jong et al., 1936-1957, cf. Ch. IV, § 4). 14. The view that phosphate colloids, and especially lecithins, are an important constituent of the plasma-membrane, finds support in the results of some preliminary experiments on the effect which other uni- and bivalent ions exercise on the speed with which water passes into the gymnoplasts. When the univalent ions are arranged according to the degree in which equal concentrations of them increase the resistance of the plasma-membrane against the passage of water, we obtain the sequence Rb < Li < Cs < Na < K, which agrees completely with that which Teunissen (1936) and Teunissen and Bungenberg de Jong (1938) found when they determined the concentrations of univalent cations that are required for reversing the electric charge of lecithin. The noxious influence of Mg- and Be-ions made it difficult to work out a similar sequence for the bivalent ions, but it probably is Be (?) < Mg (?) < Ba < Sr < Ca.