Water potential by gravimetric method:Measuring the Water Status of Potato Tubers

Measuring the Water Status of Potato Tubers

Objectives: Upon completion of this laboratory you should be able to:

1. determine the water potential of a plant tissue by the Chardokov and gravimetric
techniques and understand the underlying theory. method and understand the underlying theory.

2. determine the osmotic potential of a plant extract by the freezing point depression 3. describe techniques for measuring the pressure potential of a plant tissue. 4. describe techniques for measuring plant water status such as the pressure bomb, vapor
pressure osmometer, and psychrometer. Introduction Water potential (w, psi), which is a measure of the energy state of water is affected by dissolved solutes, pressure and matrix particles. The contribution to water potential by dissolved solutes, termed osmotic potential (s ), is always negative in sign. In other words, solutes decrease the water potential. The contribution of pressure ( p) may be positive, negative or zero, but is generally positive since most plant cells are turgid (turgor pressure). The contribution due to the binding of water to colloidal particles (matric) and surfaces, termed matric potential (m), also lowers the water potential. Although it is often small enough to be ignored, matrix potential is important when considering soil water relations. Thus, the water potential of a plant system can be arithmetically represented by the equation: w = s + p + m In this lab we will use the Chardakov and Gravimetric techniques to determine the water potential (w) of a potato tuber cells. We will determine the solute potential (s ) by the Freezing Point Depression Method. Pressure in the cells can be arithmetically calculated once s and w are known. If time permits, we will also measure the water conductivity of potato tubers, determine the Q10 for water transport into potatoes and prepare a Hofler diagram. Post-Lab Assignment

1. Write an abstract of this experiment 2. Append to your abstract the following (be sure to include appropriate captions for your
tables and graphs): Chardakov Tables 1 & 2. Calculate the water potential of the potato based on these data. Gravimetric Table 2. Plot % change in weight vs. [sucrose]; include best fit line with regression analysis data. Calculate the water potential of the potato based on these data. In your abstract discuss how the water potential values obtained from the Chardakov and Gravimetric techniques compare and which is likely most accurate. Freezing point Tables 1 & 2. Plot temperature vs. time for both water and sap on the same graph.

3. In your abstract be sure to include: the pressure (p) in the potato cells and discussion
whether the data you collected supported your hypotheses.

Chardakov Method for Determining Water Potential

Background Information: The Chardokov method provides a quick means to determine plant tissue water potentials. This method depends on the change in density in a solution that occurs after a tissue has been immersed in it. The solution gains or looses water depending on the water potential of the tissue. If the density of a solution does not change (no net movement of water) then this solution has the same water potential as the tissues that were incubated in it. It is assumed that solute movement between tissue and solution is negligible. Density changes can be observed by watching whether a drop of the original solution floats or sinks in the test solution after tissue incubation. Alternately, for a more accurate measurement of changes in the solution density, a refractometer can be used. Question: What is the water potential of potato tissue? Hypothesis: Water potentials (w) of the potato tuber cells will be negative and should range from -0.1 to -1.0 MPa (Bland and Tanner, 1985; Barcelo et al, 1994). Protocol:

1. Dispense 10 mL of water or a sucrose solution (0.1 - 0.8 molal) into each of nine 2.

3. 4. 5. 6. 7. 8. 9.
Data:

appropriately-labeled test containers (note: sorbitol, mannitol or polyethylene glycol can be used in place of sucrose). Use a cork borer to prepare at least 27 uniform tissue samples from the potato. Cut them to the same length (ca. 4.0 cm) with a razor blade and be sure not to include any fragments of the skin. Work quickly to minimize evaporation and keep the tissue wrapped in a moist towel. Put two or preferably three potato cores in each solution (water or sucrose). If necessary, add more of the appropriate solution to completely submerge the cores but the final volume in each tube must be the same. Incubate the cores for at least 1.5 h, preferably longer. Periodically swirl the containers. Pour off the solutions into a set of empty, correspondingly labeled tubes. Mix the tubes thoroughly with a vortex mixer. Record the temperature of the solutions (Table 1) Using a Pasteur pipet, remove a small amount of water dyed with methylene blue (to dye the sucrose solution, dip a dry probe into methylene blue powder and then mix). Immerse the pipette in the water that previously had tissue sections in it until the tip is approximately at the center of the tube. Slowly release a drop of the methylene blue solution from the pipette and note whether the drop of the dye sinks, disperses, or floats to the surface in this solution and subjectively estimate whether it does so rapidly or slowly. Do this gently! Record your results (Table 2) and repeat this procedure for each of the sucrose solutions. Be sure to use a different pipet for each dye stock.

1. Complete Tables 1 and 2.

Table 1: Temperature Data Temperature of the solutions in which the potato cores were incubated Temperature (C) Temperature (K)

Table 2: Response of drops (float, sink, hover) when placed in solutions in which potato cores have been incubated [Sucrose](molality) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Analysis & Conclusions: Drop Response

1. Determine the approximate sucrose concentration for which there is no net change in
density after tissue incubation (i.e., drop disperses, the w tissue = w solution). 2. Calculate s (= w) in an open system such as a solution in a beaker): s = -miRT where

m = molality (moles/1000 g) i = ionization constant (1.0 for sucrose) R = gas constant (0.00831 liter MPa mol-1 K-1 ) T = Temperature in degrees K (C + 273 = K) note: the units are MPa NOTE: the following is the preferred SI form for the equation. The answer will be in Jm -3 which are equivalent to pressure (Pa). Divide by 106 to convert to MPa. s = -miRT where

M = molality (1 molal = 1 x 103 mol m-3 H2O) i = ionization constant (1 for sucrose)

R = gas constant (8.31 JK-1 mol-1 ) T = temperature (K)

3. What is the water potential of the potato tuber cells? How does this value correlate to
the expected values? Is this technique accurate? Explain.

4. We incubated the cores for about 1.5 hours. How can we determine if this is an
adequate time?

Measuring Water Potential by the Gravimetric Technique

Background: This technique for measuring water potential is similar in theory to the Chardakov method and shares the advantage of being simple to perform and doesn't require expensive equipment. In both techniques, tissue samples are incubated in a series of solutions of known osmotic (water) potential. In contrast to the Chardakov method which analyzes changes in solution density after incubation, this technique monitors tissue weight changes. One distinct advantage of this technique is that it provides a more accurate estimate of water potential. In this method, tissue samples are weighed before and after incubation in a series of solutions of known osmotic (water) potential. Then, the percent change in weight of the tissue is plotted versus solution concentration (or osmotic potential). The water potential of the tissue is considered to be equal the osmotic potential of the incubating solution at which there is no change in tissue weight (i.e., where the curve intercepts the x-axis). A "kink" may be observed in the graph below the x-axis. This is due to incipient plasmolysis that occurs at low solution water potential. Water potential values determined by this method may be slightly more negative than those obtained by the Chardokov method. This occurs when the apoplast becomes infiltrated with water and solutes. This increases the tissue weight and may lead to small errors. Question: What is the water potential of potato tissue? Hypothesis: Water potentials (w) will be negative and should range from -0.1 to -1.0 MPa (Bland and Tanner, 1985). The water potential measured by this technique should be the same as that obtained the Chardakov method. Protocol:

1. Dispense 10 mL of water or sucrose (0.1 - 0.8 molal) into each of nine appropriately2.

3. 4. 5.

labeled containers. (note: sorbitol, mannitol or polyethylene glycol can be used in place of sucrose). Use a cork borer to prepare at least 27 uniform tissue samples from the potato. Cut them to the same length with a razor blade (ca. 4.0 cm). Be sure not to include any fragments of the skin. Work quickly to minimize evaporation and keep the tissue wrapped in a moist towel. Weigh two or preferably three cores, record your data in Table 2 and then place the cores in one of the test solutions. Repeat for all solutions. Weigh the cores to the nearest 0.01 g. If necessary, add more of the appropriate sucrose solution to completely submerge the cores but, the final volume in each tube must be the same. Incubate the cores for 1.5 - 2.0 hours. Periodically swirl.

6. After 1.5 - 2.0 hours, record the temperature of the solutions (Table 1). Then remove the

tissues, gently blot on paper towels and reweigh. Record your data in Table 2. Examine the cores as you weigh them. Describe their relative turgor (stiff/limp).

Data:

1. Complete Table 2. Use the following equation to calculate the percent change in weight
for each tissue by the following equation: % change = (final - initial)/initial x 100

2. Plot % change in weight vs. sucrose concentration (molality). Draw the best fit line for 3.

4.

your data. From the graph, determine the concentration of the sucrose solution in which there was no net weight gain (i.e., % change = 0). At this point, the water potential of the solution equals the water potential of the potato cores. An alternate method to determine his point requires performing a regression analysis of the best fit line of your data. The equation for this line is in the form, Y = mx + b. Substitute in this equation, Y = O, and then solve for X (the point at which the line crosses the X axis and equals the sucrose concentration in which there is no net change in weight of the cores = water potential of the cores). Which method do you think will be more accurate? Calculate the osmotic (= water) potential of this solution (see the Chardakov Lab for equations). Table 1: Temperature Data Temperature of the solutions in which the potato cores were incubated Temperature (C) Temperature (K)

Table 2: Change in weight of potato cores incubated in sucrose solutions [Sucrose] (molality) Initial Weight (g) Final Weight (g) Change in Weight (final - initial) (g) % Change in Weight

0 0.1 0.2 0.3 0.4 0.5 0.6

0.7 0.8 Analysis & Conclusions: What is the water potential calculated by this method? Does it correspond to the value obtained by the Chardakov technique? Which is more accurate? Explain. Do the cores show various degrees of turgor? Explain.

Determining Osmotic Potential by the Freezing Point Depression Method

Background Information: Osmotic potential (s) represents the effect of solutes on the energy state of water. Osmotic potential is related to other properties of the solution such as vapor pressure, boiling point, and freezing point. These properties, which are interrelated, are termed 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 freezing point of pure water is 0 C but the freezing point of aqueous solutions containing non-volatile dissolved solutes is less than zero. Thus, the addition of solutes depresses the freezing point of water. The extent to which the freezing point is depressed below O C is proportional to the concentration of dissolved solute particles; a 1 molal solution of an ideal non-ionized solute has an osmotic potential of -2.27 MPa and freezes at -1.86 C. Based on this relationship the osmotic potential of any unknown solution can be calculated: -2.27 MPa / -1.86 C = s / f rearranging: s = (1.22 MPa deg-1 ) f

where f is the freezing point of the solution in oC. Since this equation is for solutions at zero oC (273 K) we must correct the equation to yield our answer at room temperature by multiplying the equation by the ratio of the absolute temperatures (room temperature in K/273 K). Thus: eqn. 1: s = (1.22 MPa deg-1 ) f x (room temp in K/273 K) Freezing point depression is generally determined on tissue sap. Sap samples are prepared by freezing the tissue to rupture cells and then squeezing the frozen material by hand through cheesecloth or with a hydraulic press. Alternately, the tissue can be homogenized (without water) in a blender or with a mortar and pestle. In practice, the temperature of a sap sample is monitored with a thermocouple or sensitive thermometer as it is cooled. Data from a typical freezing point depression experiment is given below (Figure 1). Point A represents the extent of super-cooling of the solution. The line from A to B represents a very rapid freezing of the water in the solution after super-cooling. The increase is due to the release of the heat of fusion. Therefore, Point B is an estimate of the freezing point depression and is called the apparent freezing point (f'). The true freezing point (f) is obtained after correcting for super-cooling according to the following equation (Reiss, 1996): eqn. 2: f = f' - 0.0125 where f = true freezing point f' = apparent freezing point

 = degrees of supercooling (negative in sign) 0.0125 = amount of water (1/80) that solidifies per degree of supercooling One disadvantage of this method is that the solute composition of the sap influences the accuracy of the measurement. The equation above is given for ideal solutions of glucose or mannitol. Sucrose, on the other hand has a freezing point depression of 2.06 oC, not 1.86oC. Nevertheless, the method still provides an estimate in reasonable agreement with those obtained by other methods. Fig. 1. Typical freezing point curve click here for larger image Fig 2. Freezing point apparatus click here for larger image

Question: What is the osmotic potential of potato tissue? Hypothesis: Bland and Tanner (1985) report that the osmotic potential for a potato tube is in the range of -0.58 to -1.53 MPa. Our tubers should fall within that range. Protocol:

1. Set up the freezing point apparatus (Figure 2). 2. First, we will calibrate the thermometer by placing 3 mL of water in the sample test tube. 3. Immerse this unit in a beaker (600 mL) containing salt and ice. The ice bath should be 4. 5. 6.

7.

between -5 and -10 oC. Suspend a thermometer in the sample making sure that it doesn't touch the bottom or sides of the tube. Stir the sample continuously and watch the decrease in temperature. When the temperature reaches 0oC, record the temperature every 10 seconds (Table 1). Continue readings as the sap cools. Note the temperature closely. Periodically tap the thermometer gently to induce freezing. When freezing occurs there will be a rapid increase in temperature. Note this temperature which is the apparent freezing point (f') and continue to record temperatures until they become fairly constant. [In some cases, the sample may freeze without releasing a detectable heat of fusion. If this occurs, discard the sample and begin again. You can often determine that this has occurred if there is a plateau in the cooling rate of the sap.] The freezing point of the water should be zero. If the f' of the water is not 0oC, then correct the f' of the sap sample accordingly. Repeat the experiment with a potato sap sample. To prepare the potato sap, obtain frozen potato sections and carefully remove the peel . Homogenize the thawed potato in a blender. Transfer the homogenate to a centrifuge tube and spin at the highest setting for five minutes to recover the supernatant.

Data:

1. 2. 3. 4.

Complete Tables 1 & 2. Plot temperature vs. time for both sap and water. Calculate the true freezing point (f) of the sap (Table 2). Calculate the s of the sap sample. Table 1: Freezing response of water and potato sap Temperature ('C) Time (sec) Water Sap

Table 2: Summary Table - Freezing Point Depression room temperature (C) room temperature (K) f' water (from graph, C) f' sap (uncorrected; from graph, C) f' sap, corrected for thermometer, C sap - coldest temp during supercooling (from graph, C) sap - coldest temp during supercooling (corrected for thermometer; C) (degree of supercooling = f' sap - coldest temp, negative in sign) f true fp (C) s (MPa) s (MPa; corrected for room temperature)