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Most organisms are comprised of at least 70% or more water. It provides structural support via turgor pressure in plants. Some plants, like a head of lettuce, are made up of nearly 95% water. It is utilized by the cells as a solvent for the uptake and transport of materials such as molecules. The diffusion of water across a semi-permeable membrane is called osmosis. In this process, water moves from an area of higher energy to an area of lower energy. Water potential is the measure of free energy of water in a solution. Water potential (Ψ) is based on Ψs, Ψp, and Ψm, according to the following formula: Ψ = Ψs + Ψp + Ψm Water potential (Ψ) is pressure/unit volume, and is the measure of the tendency of water to move from a system to another and is expressed by MPa. Solute potential, also called osmotic potential, (Ψs) represents the effects contributed by the dissolved solutes in water; pressure potential (Ψp) represents the effects contributed by pressure, whereas matric potential (Ψm) represents the effects contributed by water-binding colloids in the cells. Potatoes (Solanum tuberosum) are important food crops in the cooler regions of the world. The tuber, the edible part of the white potato is a very short and thick, starchy stem, with the "eyes" being the buds on the stem. White potatoes have firm tissue and convenient size, so they are favorite subjects for the laboratory determination of the water potential of plant tissue. In this experiment, the group is expected to measure the water potential and osmotic potential in a tissue, and then calculate the pressure potential.

MATERIALS AND METHODS

Potato Tubers Sucrose (C12H22O11) solutions (0.1 m; 0.2m; 0.3m; 0.4m; 0.5m; 0.6m; 0.7m) Distilled water No.5 Cork borer Balances and weighing paper Plastic cups Funnel, 150 mm Paper towels

Blender Heidenhain (freezing) thermometers Potato Peelers Knife Ruler Cheesecloth

Part 1: Determination of water potential Eight different sucrose concentrations (Distilled water, 0.1m, 0.2m, 0.3m, 0.4m, 0.5m, 0.6m, 0.7m) were prepared and were dispensed separately into plastic cups. Using the cork borer, 16 cylinders were bore from a large potato and were cut into 4 cm slices. The cylinders were blotted with paper towels and were weighed in sets of two. The weight was recorded as the initial weight (Wi). One set of cylinders were placed in each of the plastic cups containing the sucrose solutions. After 30 minutes, the cylinders were removed, blotted with paper towels, and were weighed again. The weight was recorded as the final weight (Wf). The change in weight (∆W) and the percent change in weight (%∆W) were computed using the following formula: ∆W = Wf - Wi %∆W = ∆W Wf All results were tabulated. The % change in weight was plotted against sucrose concentration, the best-fit line drawn through the points. The molal concentration of sucrose that gives 0% change in weight was determined. The Ψs in bars of that sucrose solution was computed using the following formula: Ψs = -miRT

In this equation, m is the molal concentration of sucrose that gives 0% change in weight, i is the ionization costant (1 for sucrose), R is the gas constant (equal to 8.31 J K-1 mol-1), and T is the room temperature expressed in Kelvins (°C + 273). The water potential of the potato was determined under the assumption that the value of Ψ m is small and therefore negligible. Part 2: Determination of solute potential Ψs of extracted sap by cryoscopy The leftover potatoes from part 1 were chopped and pureed in a blender. The blended potato was filtered using cheesecloth, its filtrate was placed in a beaker. The temperature of the crushed ice-salt bath was obtained by immersing the heidenhain thermometer. The reading was recorded. 60 mL of the sap was placed in a beaker. The thermometer was inserted into the beaker and was placed into the ice-salt bath, stirring vigorously. When the temperature read 1°C, the temperature was read and recorded every 10 seconds. A plot of the temperature versus time was made.

RESULTS AND DISCUSSION In the cups containing distilled water 0.1, and 0.2 molal sucrose solutions, the potatoes increased in weight because water moved in due to the potato’s lower water potential with respect to the solution. Hence, the percent change in mass is positive. Sucrose Concentration 0 (Dist.H20) 0.1 m 0.2 m 0.3 m 0.4 m 0.5 m 0.6 m 0.7 m Initial Weight (in grams) 3.3660 3.3493 3.7560 3.4850 3.8923 3.9682 3.5191 3.5153 Final Weight (in grams) 3.5856 3.5185 3.8711 3.4440 3.6653 3.6555 3.0111 2.9189 Change in Weight 0.2196 0.1692 0.1151 -0.0410 -0.2270 -0.3127 -0.5080 -0.5964 % Change in Weight 6.12 4.81 2.97 -1.19 -6.19 -8.55 -16.87 -20.43

In the cups containing 0.3 - 0.7 molal sucrose solution, the potatoes decreased in weight because water moved out due to the potato’s higher water potential with respect to the solution. Hence the percent change in mass is negative.

Figure 1 Percent Change in Weight vs. Sucrose Concentration

The graph depicting the percent weight change (y) as a function of the sucrose concentration (x) is shown in Figure 1. Because none of the given solutions gave a 0% change in weight, the equation of the best-fit line (Figure 1) was used to solve for the exact molal concentration needed to produce the desired effect. Hence,

The equation of the best-fit line: If percent weight change is 0:

y = -39.494x + 8.9892 0 = -39.494x + 8.9892

-8.9892 = -39.494x x = -8.9892-39.494 x = 0.22760 ≈ 0.23 Hence, if a potato cylinder is immersed in a 0.23 molal sucrose solution, the potatoes will retain the same weight because the water moving out will be balanced by the water moving into the potato cells since the solution’s water potential is equal to the potatoes’ water potential. Knowing Ψp=0MPa in a free standing solution and Ψm is neglected, then Ψ = Ψs, the water potential of the potato can be solved by the following equation: Ψs = -miRT =(-0.23 molal x 1 x 103 mol m-3) (1) (8.31 J K-1 mol-1) (25 + 273K) = -569 567.4 J/m3 / 106 Ψs = - 0.57 MPa

Solute potential (Ψs) represents the effect of solutes on the energy state of water. Solute potential is related to other properties of the solution such as vapor pressure, boiling point, and freezing point. These properties, which are interrelated, are called colligative properties, and are dependent on the mole fraction of solute. Since these properties are inter-related, one can be measured and used to calculate the others. The cryoscopic osmometer measures the osmotic potential of a solution by measuring its freezing point. Solutions have colligative properties that collectively depend on the number of dissolved particles and not on the nature of the solute. One of the colligative properties of solutions is the decrease in the freezing point as the solute concentration increases. A 1 molal solution of an ideal non-ionized solute has an solute potential of -2.27 MPa and freezes at -1.86 C. Based on this relationship the solute potential of any unknown solution can be calculated: Ψs = (1.22 MPa deg-1 ) Tf, where Tf is the freezing point of the solution in oC.

Since this equation is for solutions at zero oC (273 K) the equation must be corrected to obtain the answer at room temperature by multiplying the equation by the correction factor which is the ratio of the absolute temperatures (room temperature in K/273 K). All in all: Ψs = (1.22 MPa deg-1 ) Tf x (room temp in K/273 K)

Figure 2 Temperature versus time graph of the extracted sap

In a typical freezing point curve, there is a continuous drop in temperature as the liquid is cooled, then it remains constant signifying that both liquid and solid phases are present. Temperature finally drops again when only solid phase is present. From the temperature vs. time curve shown above, the temperature became stable for thirty seconds at -3.6°C. This will be the designated freezing point for the sap extract. However, this is merely an estimate and is called the apparent freezing point. The true freezing point (Tf) is obtained after correcting for super-cooling according to the following equation:

Tf = Tf' - 0.0125 ts ts = Lowest temperature - Tf' Where Tf is the true freezing point and Tf' is the apparent freezing point. ts is the degrees of supercooling (negative in sign), while 0.0125 corresponds to the amount of water (1/80) that solidifies per degree of supercooling. Tf = Tf' - 0.0125 ts = -3.6 – 0.0125 (-3.7 - -3.6) = -3.6 – (-0.00125) = -3.59875 ≈ -3.6 Correction also has to be made for the zero point of the thermometer to determine the solute potential. Tf = Tf' - 0.0125 ts = 0 – 0.0125 (-3.7 – 0) = -0.0463 ≈ -0.05 The solute potential at 0°C can now be determined: Ψs = (1.22 MPa deg-1) Tf x (temp in K/273 K) = (1.22) (-3.6) °C x [(0+273)/273 K] = -4.392 MPa At room temperature however, Ψs = (1.22 MPa deg-1) Tf x (temp in K/273 K) = (1.22) (-3.6) °C x [(25+273)/273 K] = -4.794 MPa

Lastly, the pressure potential (Ψp) of the cells of the potato can be solved by looking back at the formula for water potential: Ψ = Ψs + Ψp (Ψm is neglected) Under equilibrium conditions, the water potential of the potato is equal to the water potential of the 0.23 molal sucrose solution. Therefore, the water potential (Ψ) of the potato is -0.57 MPa. The computed solute potential of the potato sap extract at room temperature is -4.794 MPa.

Ψ = Ψs + Ψp -0.57 MPa = -4.794 MPa + Ψp Ψp = 4.794 MPa - -0.57 MPa Ψp = 4.224 MPa

REFERENCES Journals: Bland, W. L. and C. P. Tanner. 1985. Measurement of the water potential of stored potato-tubers. Plant Physiology 79: 891-895. Boyer, J. S. 1969. Water status measurements in plants. Annual Reviews of Plant Physiology 20: 351-364. Books: Meyer, B. S. and D. B. Anderson. 1935. Laboratory Plant Physiology. Edwards Bros., Ann Arbor. 107 pp. Salisbury, F. B. and C. W. Ross. 1992. Plant Physiology, 4th Ed. Wadsworth Publishing Co., Belmont, CA. 682 pp. Website: Saupe, S. G. (2009, January 7). Determining Osmotic Potential by the Freezing Point Depression Method. Retrieved November 22, 2010, from Plant Physiology (Biology 327) Home Page: http://employees.csbsju.edu/SSAUPE/biol327/Lab/water/water-lab-freez.htm

EXPERIMENT 1

CELL WATER POTENTIAL

SUBMITTED BY: GROUP No. 8 Buenaflor, Maria Katrina A. De Leon, Eugene Morada, Jayvee V. Sison, Marcus Isaiah

SUBMITTED TO: Mr. Josefino Castillo

DATE SUBMITTED: November 25, 2010

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