Grafts et al. — 70 — Water in Plants 



vacuolar contents and the existence of phenomena associated with the col- 

 loidal state. 



In addition to osmotic water, certain authors suggest that water may 

 be held in vacuoles by entirely different mechanisms in which metabolic 

 energy plays a dominant part {cf. Bennet-Clark, Greenwood, and 

 Barker, 1936; van Overbeek, 1942). These theories, involving so-called 

 "active" water relations, will be discussed in Qiapter VIII. 



Methods for Determining Water Partition:— No method has been devised by which 

 the relative amounts of water in cell wall, protoplasm, and vacuole may be determined 

 accurately. A common method has been the expression of sap from whole tissues, 

 either in the living condition or after killing by freezing, boiling, or treatment with 

 ether, etc. The fluid thus obtained must be termed whole cell sap since it is a com- 

 posite liquid having its origin in all three cellular phases. That such sap is a true or 

 representative sample of vacuolar contents is erroneous even though, for tissues com- 

 posed of mature vacuolated cells it may consist predominately of vacuolar liquid. Sap 

 expression techniques will be considered in Chapter VII. 



Perhaps the most reliable information on the composition of vacuolar sap has been 

 obtained by the use of large cells of certain algae. Nitella. with cells up to 1^/^ inches 

 long and % inch in diameter, has been used; Valonia, a large spherical coenocyte, is 

 even more convenient. These large cells may be broken open and slightly compressed 

 whereupon from a drop to a cubic centimeter or more of sap is obtained. This repre- 

 sents a fairly reliable sample of the vacuolar sap of such plants, and many experiments 

 dealing with permeability and solute absorption have been made using these cells. Re- 

 cent studies have involved absorption of radioactive isotopes and accurate measures 

 of the partition of these elements have been obtained. 



Micrurgical techniques have been devised {see for example Livingston and Dug- 

 gar, 1934) whereby rather pure vacuolar sap may be secured from even small cells, 

 e.g., hair cells of tobacco, by inserting a small hollow needle into the vacuole and draw- 

 ing out the liquid. The method is laborious and provides very small samples that re- 

 quire analysis by micro methods. 



Chibnall (1923) treated spinach leaves with ether as a means of separating 

 vacuolar from protoplasmic sap. The cells rapidly plasmolyzed, the vacuoles mark- 

 edly decreased in volume, and a clear liquid could be obtained on applying pressure. 

 Examination showed no rupture of cells, and the protoplasm remained essentially in- 

 tact. He admitted that the "vacuolar" liquid contained some protoplasmic constituents, 

 but believed that the expressed sap was almost entirely vacuolar in origin. The ground, 

 pressed residue gave a measure of the protoplasmic and wall sap. 



Mason and Phillis (1939) estimated the partition of water between the vacuole 

 and protoplasm of the cotton leaf in two ways. First, by determining the concentra- 

 tion of chlorine and potassium in press sap assumed to be vacuolar in origin, in sap 

 assumed to be protoplasmic in origin, and in whole cell sap expressed from tissue that 

 had been frozen. They calculated the amount of water associated with the quantities 

 of CI" and K* found. Vacuolar sap they designate as that expressed from living leaves 

 where shearing forces are avoided. Protoplasmic sap they take as that originating 

 from the frozen and thawed press cake remaining after expression of vacuolar sap. 

 Second, by plotting the weight of sap (believed to be vacuolar) expressed from living 

 leaves against pressure applied in uniform increments. The total weight of vacuolar 

 sap was calculated by extrapolation. 



The authors concluded that only about 30 per cent of the total cell water is present 

 in the vacuoles of cotton leaves. This value seems low and both procedures for its 

 calculation are subject to criticism. The possibility of water in amount disproportional 

 to solute expressed from cell walls and protoplasm, due to the behavior of cell colloids 

 toward water and dissolved substances at the higher pressures seems likely. It also 

 appears certain that some filtration of solutes by the cytoplasm would occur when liv- 

 ing cells are submitted to pressure. The fact that solute concentration of the vacuolar 

 sap was only one-fifth that of protoplasmic sap could be explained in this way. Be- 

 cause residues from expression of living leaves absorbed water, even from protoplasmic 

 sap, it seems that the pressures used must have brought about a separation of water 

 from the solutes in the cells, giving a false measure of the concentration of the so- 

 called "vacuolar" sap. 



Determination of the volume of each cellular phase will give a rough indication of 

 the partition of water in the cell ; there would be little error in assuming that the vol- 



