Transpiration Inquiry Problem: Design an experiment to quantify the effect of Humidity, Temperature, Wind, Light Intensity or other logical variable on the rate of transpiration in a small plant. Background: The amount of water needed daily by plants for growth and maintenance of tissues is small in comparison to the amount that is lost through the process of transpiration (the evaporation of water from the plant surface) and guttation (the loss of liquids from the ends of vascular tissues at the margins of the leaves). If this water is not replaced, the plant will wilt and may die. The transport of water up from the roots in the xylem is governed by differences in water potential (the potential energy of water molecules). These differences account for water movement from cell to cell and over long distances in the plant. Gravity, pressure, and solute concentration all contribute to water potential, and water always moves from an area of high water potential to an area of low water potential. The movement itself is facilitated by osmosis, root pressure, and adhesion and cohesion of water molecules. The Overall Process: Minerals actively transported into the root accumulate in the xylem, increasing solute concentration and decreasing water potential. Water moves in by osmosis. As water enters the xylem, it forces fluid up the xylem due to hydrostatic root pressure. But this pressure can only move fluid a short distance. The most significant force moving the water and dissolved minerals in the xylem is the upward pull as a result of transpiration, which creates tension. The “pull” on the water from transpiration results from the cohesion and adhesion of the water molecules. The Details: Transpiration begins with evaporation of water through stomates (stomata), small openings in the leaf surface which open into air spaces that surround the mesophyll cells of the leaf. The moist air in these spaces has a higher water potential than the outside air, and water tends to evaporate from the leaf surface (moving from an area of high water potential to an area of lower water potential). The moisture in the air spaces is replaced by water from the adjacent mesophyll cells, lowering their water potential (since the cytoplasm becomes more concentrated). Water will then move into the mesophyll cells by osmosis from surrounding cells with higher water potentials, including the xylem. As each water molecule moves into a mesophyll cell, it exerts a pull on the column of water molecules existing in the xylem all the way from the leaves to the roots. This transpirational pull occurs because of (1) the cohesion of water molecules to one another due to hydrogen bond formation, and (2) by adhesion of water molecules to the walls of the xylem cells which aids in offsetting the downward pull of gravity. The upward transpirational pull on the fluid in the xylem causes a tension (negative pressure) to form in the xylem, pulling the walls of the xylem inward. The tension also contributes to the lowering of water potential in the xylem. This decrease in water potential, transmitted all the way from the leaf to the roots, causes water to move inward from the soil, across the cortex of the root, and into the xylem. Evaporation through the open stomates is a major route of water loss in plants. However, the stomates must open to allow the entry of CO2 used in photosynthesis. Therefore, a balance must be maintained between the gain of CO2 and the loss of water by regulating the opening and closing of stomates on the leaf surface. Many environmental conditions influence the opening and closing of stomates and also affect the rate of transpiration. Temperature, light intensity, air currents, and humidity are some of these factors. Different plants also vary in the rate of transpiration and in the regulation of stomatal opening.

Materials: Two small potted plants Balances Plastic bags or parafilm String or Tape Misting bottle Greenhouse or Grow Box Fan Note: Not all materials will be used by every group. “Whole-Plant” Transpiration Assay: For this lab, you will use a method of assaying transpiration that is referred to as the “wholeplant” method. The general details for the whole-plant method are as follows: 1. Obtain two small potted plants. All of the plants you use should be the same species. 2. Remove each plant from its pot. The roots should remain intact. Do not attempt to remove soil from the roots. However, it is OK if some soil remains in the pot. 3. Remove all blooms/flowers from the plants. 4. Enclose the entire root ball of each plant in a plastic bag or wrap in parafilm. Make sure that there are no holes in the bags or gaps in the parafilm. Tie each bag snugly around the stem with string or, if using parafilm, be sure the parafilm is wrapped around the stem (without breaking the stem). It is imperative that the bag is snug around the stem so that as little water as possible can evaporate out at that location. 5. Clearly label each plant with your name(s) and the condition that the plant will be exposed to. Along with the “normal room” conditions, you will test one condition: under constant bright light, high temperature, a fan set at low, in the dark, high humidity, or any other variable you can think of (be sure to get instructors approval). 6. Determine the mass of each plant and record it in your data table. 7. Put each plant in its appropriate condition. 8. Place a thermometer in each condition. Record the temperature of each location. 9. Return each day to take the mass of each plant. Try to return at approximately the same time of day if possible. Record all mass measurements. If your plant blooms, pinch the blooms off and be sure any leaves or blooms that fall off are put back in the center of the plant to be included in the total mass of the plant. 10. You should also record the temperature at each location each day. 11. Continue taking measurements until the end of the week. Steps to complete before beginning your experiment: Develop a detailed protocol & clear experimental plan for your experiment. Develop a data table for your experiment. Determine any calculations that will be necessary for your data. Think about how you will graph your results. After reviewing your protocol and discussing any safety hazards, your instructor must approve your protocol before you can conduct your experiment.

Data Collaboration: Following the completion of the lab, you will collaborate with other groups to share your data. Your lab report must include your individual data, class data, and a graph showing the % change in mass for plants in each experimental condition and the normal room control (average values for conditions that had more than one plant are acceptable (and recommended for your sanity!). Analysis Questions (to be answered in your report following your conclusion): 1. For this experiment, what was the independent variable and what was the dependent variable? What were the constants? 2. Calculate the average rate of water loss per hour for each of the treatments. In order to do this, you need to know that 1 mL of water has a mass of 1 gram. Express your answers in mL/hr. 3. For each of the non-control conditions, give the effect the condition had on transpiration, and then explain why it had that effect. 4. Do you think temperature had an impact on your experiment? Explain your answer. 5. Explain the role of water potential in the movement of water from soil through the plant and into the air. 6. What is the advantage to a plant of closed stomata when water is in short supply? What are the disadvantages? 7. Describe several adaptations that enable plants to reduce water loss from their leaves. Include both structural and physiological adaptation. You may need to do some research to answer this question. 8. Why did you need to calculate the % water loss each day instead of graphing the total

amount of water lost each day?

Counting Leaf Stomata Introduction: Plants and animals both have a layer of tissue called the epidermal layer. Plants have special pores called stomata to allow passage of material. The stomata pores are surrounded on both sides by jellybean shaped cells called guard cells. Unlike other plant epidermal cells, the guard cells contain chlorophyll to do photosynthesis. This allows the cells to expand/ contract to open or close the stomata. Guard cells also close when dehydrated. This keeps water in the plant from escaping. The opening or closing of guard cells can be viewed in a microscope by adding different water concentration to the leaf tissue. Most stomata are on the lower epidermis of the leaves on plants (bottom of the leaf). The number of stomata on the epidermal surface can tell you a lot about a plant. Usually, a high concentration of stomata indicates fast growth and wet climate. Lower concentrations of stomata indicate lower rates of photosynthesis and growth or adaptations for dry weather. Purpose: To view and compare the stomata from the leaves of several species of plant Materials: 3 leaves (1 from 3 different species) Compound light microscope 3 microscope slides Clear nail polish Transparent tape Procedure: 1. 2. 3. 4. 5. 6. 7. 8. 9.

Obtain three leaves from different types of plants. Paint a thick patch (at least one square centimeter) of clear nail polish on the underside of the leaf surface being studied. Allow the nail polish to dry completely. Tape a piece of clear cellophane tape to the dried nail polish patch. Gently peel the nail polish patch from the leaf by pulling on a corner of the tape and "peeling" the fingernail polish off the leaf. This is the leaf impression you will examine. Tape your peeled impression to a very clean microscope slide. Use scissors to trim away any excess tape. Label the slide with plant name. Examine the leaf impression under a light microscope at 400X. Search for areas where there are numerous stomata, and where there are no dirt, thumb prints, damaged areas, or large leaf veins. Draw the leaf surface with stomata. Count all the stomata in one microscopic field. Record the number on your data table.

10. Repeat counts for at least three other distinct microscopic fields. Record all the counts. Determine an average number per microscopic field. 11. From the average number/400X microscopic field, calculate the stomata per mm2 by multiplying by 8. 12. Follow procedures 2 - 11 with the other leaves. Data: Leaf 1

Leaf 2

Leaf 3

Name of Leaf Drawing in 400x (with several stomata) Stomata in field 1 Stomata in field 2 Stomata in field 3 Average Stomata in field Stomata/ mm2 Analysis Questions Part II (to be answered in your report following your conclusion): 1. Which leaf had the most stomata? Why do you think this was so? 2. Explain, in detail, how guard cells open and close stomata? 3. At what time of day would stomata be closed and why? 4. Why does the lower epidermis have more stomata than the upper epidermis of a leaf? 5. Define transpiration. 6. What two gases move in and out of the leaf stomata? 7. What does a larger number of leaf stomata indicate about the growing climate of that plant?

8. Would you expect CAM plants to have as many stomata? Why or why not?