BIO 322/122L Laboratory Plant Water Relations I. Water Potential. The cytoplasm of the plant cell, with its enclosed vacuole, is contained within a membrane that is more permeable to water than to most solutes. The water potential of a cell relative to that of the surrounding solution determines whether water will move into or out of the cell. Water potential can be described mathematically as the sum of the osmotic potential and the pressure potential. R = R B+RC The osmotic potential (R ) is a function of the dissolved solute concentration (see equation B below), and it tends to pull water into the cell via osmosis. In opposition to this force is the pressure potential (R ), which equals the pressure of the cell wall and membrane on the cell D contents. While the osmotic potential is always negative, the pressure potential may be positive (pressure) or negative (tension), but is usually positive. If a cell is placed in a solution which has a R that is higher than that of the cell, there will be a net movement of water into the cell. However, if the surrounding solution has a lower ø than in the cell, there will be a net movement of water out of the cell. If this latter situation continues, the plasma membrane and cytoplasm will pull away from the cell wall, a condition known as plasmolysis. By trial and error, a concentration of bathing solution can be found that just produces plasmolysis, and this is known as incipient plasmolysis. Thus, "incipient plasmolysis" is defined as when ~50% of the cells are plasmolyzed. At incipient plasmolysis, there is no longer a pressure potential exerted by the wall (i.e. ø =0), ñ and therefore, under that condition, ø=ø. It should also be noted that for solutions, ø = ø. A solution which just causes incipient plasmolysis thus has a water potential, and, by inference, osmotic potential that is similar to the water potential (and osmotic potential) of the cell cytoplasm. Finally, since the cells we use are highly vacuolated, it can also be assumed that the osmotic potential of the cell is basically the vacuolar osmotic potential. A. Measurement of osmotic potential (ø ) by incipient plasmolysis 1. Solutions of sucrose of the molalities, 0.5, 0., 0.3, 0.25, 0.2, 0.15, 0.1 m have been prepared for you. Place a few drops of each sucrose solution in the depressions of a spot plate, one concentration to a depression. 2. Peel the epidermis along the midrib of the underside of a leaf of Rhoeo discolor, preferably using the middle part of the leaf. 3. Cut the peeled portion into small pieces and drop a piece into each depression containing a sucrose solution. Cover with a cover slip. 1
. Examine each tissue immediately under a microscope, and then after 30-60 min. The water soluble pigment in the cytoplasm of the cells makes the determination of plasmolysis relatively simple. 5. Determine the concentration which causes incipient plasmolysis (as defined above). 6. Use the following equation to convert the molality of the solution in (5) to atmospheres of osmotic potential (ø ): -ø = mirt where: m = molality of the solution i = ionization constant for the solution (for sucrose this is 1) R = the gas constant = 22. liter atmospheres/mole degree 273 T = absolute temperature (Abs Temp) -ø = molality x 22. x Abs Temp 273 0 OR: ø = molality x (-0.082) x (273 + Temp in C) B. Measurement of water potential by the Chardakov Method The Chardakov method provides a simple, and relatively inexpensive method for estimating tissue water potential. It can also easily be used for field measurements, in contrast with some other methods. In this experiment, the water potential of potato tubers will be estimated. The method depends on evaluating changes in the densities of known solutions after an experimental tissue has been immersed in them (and thereby allowed to absorb or lose water by osmosis). If the density of the solution that the tissue is immersed in does not change (i.e. there was no net water exchange), then that solution has the same water potential as the tissue. It is assumed that solute movement between the tissue and surrounding solution is negligible. Procedure 1. Label 3 sets of seven 20-30 ml test tubes as follows: 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, and 0.0 molal (m). 2. Add 10 ml of the sucrose solutions of the appropriate concentrations to two sets of the tubes, leaving the third set empty. 3. With a to 5 mm diameter (medium) cork borer, cut 15 potato sections at least 0 mm long from half of a large tuber.. Using a razor blade, trim each section to 0 mm long. Place the trimmed sections in a small 2
beaker, and keep it covered to minimize evaporation from the tissue surfaces. 5. Place three tissue sections in each of the tubes of one set of the sucrose solutions. Make sure the sections are completely immersed. 6. To each tube of the second set of solutions, add a small portion of methylene blue powder, and stir it into the solution. (The proper amount of methylene blue to use in each tube can be obtained as follows: Dip a dry dissecting needle into the dye powder; the number of crystals adhering to the needle is sufficient to make an adequately colored solution when dissolved.) 7. Keep both sets of solutions at the same temperature. After the potato sections have had a chance to lose or gain water (30 min or more, equilibrium need not be established), pour the solutions off into the third set of empty, correspondingly labeled tubes. 8. Using a disposable Pasteur pipet, carefully and slowly release a drop of the methylene bluesucrose solution from the 0.15 m tube about halfway down into the 0.15 m sucrose solution that had previously bathed the potato sections (see Figure below). Note whether the drop of dye-sucrose solution sinks or floats in the solution, and subjectively estimate whether it does so rapidly or slowly. Record your observation. 9. Repeat the procedure in (8) for the other dye-sucrose and corresponding sucrose solutions, and record the result for each. 10 Estimate, by interpolation if necessary, the molality of the dye-sucrose solution which would neither sink nor float up to the surface (in the corresponding sucrose solution). This solution must have the same water potential as the tissue, since it has the same density as the sucrose solution that had bathed the potato sections (and which did not lose or gain water). 3
II. Permeability of Living Tissues to Acids and Bases In general, cell membranes are more permeable to uncharged molecules than to charged molecules. This is mainly because of the uncharged, hydrophobic lipid core of the cellular bilayer membranes. Solutions of certain acids and bases provide convenient sources of charged and uncharged molecules (that are also small enough to penetrate membranes). In addition, there are certain pigments in plant cells that change color as a function of ph. Thus, in this experiment, you will compare the permeability of plant cell membranes to a weak acid (acetic) versus a strong acid (HCl), and also compare a weak base (NHOH) to a strong base (KOH). Remember from your chemistry that when a weak acid (or weak base) is added to water, only a portion of the molecules dissociate. However, when a strong acid (or strong base) is added to water, it can be considered that essentially all of the molecules dissociate. Materials required: a. Leaves of healthy Rhoeo plants b. Syracuse dishes or watch glasses c. Solutions of the following types (please use 5 ml. or less of each): 0.025 N HCl 0.025 N acetic acid (CH COOH) 3 0.025 N NH OH 0.025 N KOH d. Pipettes and tweezers Procedure 1. Prepare at least 8 strips of lower epidermis of Rhoeo, floating them on distilled water. 2. Place two pieces of Rhoeo epidermis into the KOH solution, and six into the NH OH solution. Record the time. Using the microscope, observe and record the time it takes for the strips to change color in each basic solution. 3. When blue coloration is achieved in all the strips, remove two pieces from the NH OH solution, briefly rinse them in water, and transfer them to acetic acid. Record the time required to change color.. Similarly, transfer two pieces from the NH OH solution to the HCl solution, and determine the time required for the strips to change color. 5. When the color change is completed, transfer the strips, now in acid, back to NH OH, and again record the time required for each to change color.
Format for the Write-up for the Water Relations Lab. This section describes what you are expected to put in your lab writeup. You may write your answers/results in this format, corresponding to these letters and numbers. You are encouraged to discuss the lab exercise with your groupmates, cooperate during lab, etc.; however, you must write up your results individually. A. Define the terms osmotic potential, pressure potential and water potential. B. Measurement of osmotic potential (Rhoeo epidermis experiment) 1. Report your observations for each concentration of sucrose. 2. What concentration resulted in incipient plasmolysis and how did you know when it occurred? 3. Based on the above, what was the osmotic potential of the cells? (Show your calculation). What were possible sources of error in this experiment? C. Measurement of water potential (potato experiment) 1. Report your observations with the Chardakov water potential measurement method; how did the drop of methylene blue-sucrose solution move (or not) in each solution? 2. Based on the above, estimate the molality of solution that would have the same water potential as the potato tuber (i.e. estimate the water potential of the tissue). D. Permeability of plant cells to acids and bases (Rhoeo leaves) 1. Report your experimental data. You may wish to make a chart listing the treatments (e.g. exposure to KOH, change from NHOH to CH3COOH, etc.), the color changes, and the time required for each, in subsequent columns. 2. Which alter cellular ph faster, weak acids and weak bases, or strong acids and strong bases? Please explain your answer. 3. The pigment that changes color is probably located in the vacuole. How many membranes must these acids and bases cross to affect this pigment? Please name them. 5