Materials Included In Kit

Additional Materials Required

Prelab Preparation

1. Use forceps or a dissecting needle to remove the cotton plugs from the pipets.
2. Use scissors or a knife to cut the tubing into 16" lengths.
3. Begin bean seeds at least two weeks in advance.
   1. Soak the red bean seeds in warm water for one hour prior to planting
   2. Place peat pellets into tray and add warm water to expand them to 1–1½" tall.
   3. Gently pour off the excess water.
   4. Pull the netting open on top of the peat pellets.
   5. Sow 2–3 seeds per pellet by pushing them under the top layer of peat.
   6. Cover the tray with the clear cover and place the tray in a warm location. Note: The optimal soil temperature for the germination of bean seeds is 65–85 °F. If the seeds start to mold, remove the cover for a day to allow better air circulation around the seeds. If the mold continues, remove the seeds and begin new seedlings.
   7. When the seeds begin to sprout, remove the cover and place the tray in a sunny location or under a grow light.
   8. Rotate the tray ¼ turn daily and water when the peat pellets turn light brown.
4. Set up the treatments areas such that they will not interfere with each other. Make labels for each treatment area placing the labels in the treatment area.
   1. Strong Light: (one of the following)
      1. Place a 1-L beaker filled with tap water between a 150-W flood lamp and the area in which students will place their samples. The water serves as a heat sink for the light so that the leaves do not become heated.
      2. Turn on a bright overhead projector and point the light directly at the area in which students will place their samples. The overhead projector should be located close to the plant area to ensure a very bright light source.
         {10797_Preparation_Figure_7}
      3. Turn on a bright overhead projector and place the potometer on the transparency area of the overhead projector.
5. Gentle Breeze:

   In a separate area place a fan on the lowest setting.

6. High Humidity:

   In a separate area place the spray bottle and clear plastic bag.

7. Room Conditions:

   Place the “Room Treatment” label in a separate area. This area should have the same amount of light as the Gentle Breeze and High Humidity areas. The room area should not have any significant breezes that would affect the transpiration rates of the plants.

Safety Precautions

The scalpel is a sharp object; care must be taken when cutting with the scalpel, always cut away from the body and away from others. Although the materials in this lab activity are nonhazardous, follow normal safety precautions. Remind students to wash hands thoroughly with soap and water before leaving the laboratory.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. Scalpels may be disposed of according to Flinn Biological Waste Disposal Method V, sharps and broken glass. All other materials in this laboratory may be disposed of using Flinn Biological Waste Disposal Method VI, in the regular trash. 

Lab Hints

• Enough materials are provided in this kit for 3 groups of students. This laboratory activity can reasonably be completed in one 50-minute class period. The laboratory can be read before coming to lab, and the data compilation and calculations can be completed after the lab.
• Ensure students label all materials with their group number.
• Assembling a bubble-free potometer takes practice; ensure that more than eight seedlings are available.
• Using a blunt-end syringe, add one drop of food coloring to the top of the water in the pipet once the potometer is assembled. The drop of dye will help the students read the amount of water lost during the activity.
• Discuss with students how scientists cope with faulty data.

Sample Data

Assigned Treatment ___room conditions___

Table 1. Potometer Readings

Table 2. Water Loss (mL/m²)

Answers to Questions

1. For each treatment, graph the class average cumulative water loss for each treatment.
   {10797_Answers_Figure_9}
   1. The independent variable: Time (minutes)
   2. The dependent variable: Water Loss (mL/m²)
2. Calculate the rate of water loss (in mL/min/m²) for each of the treatments.
   1. Room Conditions: 0.102
   2. Gentle Breeze: 0.889
   3. High Humidity: 0.081
   4. Strong Light: 0.664
3. Explain why each of the treatments caused an increase or decrease in transpiration compared with the room conditions.
   1. Gentle Breeze: An increase in wind speed results in an increase in the rate of leaf water loss because the water evaporates more rapidly off the leaf creating lower water potential in the surrounding air and therefore a greater rate of transpiration. A strong breeze may cause the stomata to close, reducing the rate of transpiration.
   2. High Humidity: Increased humidity in the air surrounding the leaf decreases the water potential gradient between the saturated air in the leaf air spaces and the air surrounding the leaf, resulting in a decreased rate of leaf water loss.
   3. Strong Light: Absorption of light results in an increase in leaf temperature; since the rate of water evaporation increases as the temperature increases, the increase in leaf temperature results in an increased rate of leaf water loss.
4. Why was it necessary to calculate the leaf surface area before calculating the water loss for each treatment?

   Each plant has a different leaf surface area. In order to compare results the amount of water lost must be adjusted (normalized) to reflect these differences.

5. Explain the role of water potential in the movement of water from soil through the plant and into the air.

   At each step of transpiration water moves from an area of higher water potential to an area of lower water potential. Students may give details in various plant structures: root hair, xylem, leaf mesophyll, leaf air space and stomata.

6. What is the advantage of closed stomata to a plant when water is in short supply? What are the disadvantages?

   The advantage to closing stomata when water is in short supply is that the closed stomata do not lose water as rapidly, preventing wilting and eventually death. The disadvantage to closed stomata is that the amount of carbon dioxide available for photosynthesis is limited and the plant cannot grow.

7. Describe several adaptations that enable plants to reduce water loss from their leaves. Include both structural and physiological adaptations.

   Adaptations to reduce leaf water loss include a thicker cuticle, fewer stomata, fewer leaves, less surface area per leaf, sunken stomata and C-4 or CAM photosynthesis.

References

Biology: Lab Manual; College Entrance Examination Board: 2001.

Introduction

In actively growing plants, tissues (leaves and root tips) can be 80–90% water while the woody parts are 45–60% water. What role does water play in plants? Why do plants need so much water to stay alive?

Objectives
After doing this laboratory, you should be able to:

• Test the effects of environmental variables on rates of transpiration using a controlled experiment.
• Calculate the rate of water lost by a plant through transpiration.

Concepts

• Transpiration
• Water potential
• Guttation

Background

Plants use water as a medium for transporting nutrients within the plant, to provide hydrostatic support and evaporative cooling, and as a reactant in biochemical processes. Water has many unique chemical properties that make it an excellent solvent. Water is a polar compound that can dissolve both ionic and polar substances such as minerals and carbohydrates, respectively. These nutrients are then transported throughout the plant. Water also acts as a reactant in many chemical reactions in the plant, including photosynthesis. In photosynthesis, water serves as the source of electrons for the conversion of carbon dioxide (CO₂) into glucose. However, only a small amount of water is needed for the actual electron transfer step in photosynthesis. Most of the water needed by a plant for photosynthesis is used to keep the cell surfaces moist so that the cells can absorb carbon dioxide gas from the atmosphere. Carbon dioxide is very soluble in water, whether it is found inside a cell (cytoplasm) or outside of the cell.

Another important function of water is that it maintains turgidity (or pressure) in plant tissue. Water literally “inflates” leaves, giving structure and support to leaves. Turgidity is also necessary for cell growth and enlargement. New cells expand in size by creating more cytoplasm, which is composed mostly of water. In fact, the loss of water is easy to see when a plant wilts. Wilting is actually the dehydration of plant cells. A plant that suffers from long term dehydration stops growing and eventually dies.

Water must follow the laws of thermodynamics. Consequently, water always moves from regions of high energy to regions of low energy. In a plant this means that water flows from regions of high water potential to regions of low water potential. This occurs through the processes of osmosis, root pressure, and adhesion and cohesion of water molecules. Water is transported from cell to cell within the plant because of differences in water potential within the plant. Water potential is actually a measure of the “free energy” of water. Free energy is defined as the total amount of energy in a physical system which can be converted to do work. Pure water has a high amount of free energy but, by convention, is defined as having a water potential of zero. Plant water potential is equal to the sum of the osmotic potential (ψ) and the turgor potential (ψ).

Osmotic potential is a measure of the amount of solutes (dissolved minerals and other nutrients) in the water within the plant. The root cells expend energy to actively transport dissolved minerals into the root. An increased solute concentration in the roots causes a lower amount of free energy and therefore a negative water potential in the root tips. Water then flows by osmosis from the region of high energy in the soil to the region of low energy in between the root cells. Osmotic potential is always negative in a plant.

Turgor potential is also known as the pressure potential. Turgor potential occurs when water molecules enter a cell and apply pressure to the cell walls. Living plant cells have positive turgor potential. The cells in wilted leaves have zero turgor potential. Specialized water transport cells called xylem have a negative turgor pressure because water is removed from xylem by the adjacent cells due to osmosis.

Plants need water around their roots consistently because they constantly lose water through their leaves via transpiration and guttation. Transpiration is the loss of water by evaporation from the leaves and is the main method for pulling water from the roots to the leaves. Guttation is the appearance of drops of sap on the tips or edges of leaves of some vascular plants. Water accumulating in the roots overnight increases the turgor potential in the leaves, forcing sap out of the tips of the leaves (see Figure 1).

Transpiration begins with evaporation of water through the stomata (singular: stoma or stomate). Stomata are tiny openings (pores) used for the absorption of CO₂ for photosynthesis and oxygen (O₂) for cell respiration (see Figure 2). Thousands of stomata occur on the underside of a typical dicot or on the upper surface of a plant whose leaves float on water. Each stoma is formed by a pair of specialized cells known as guard cells which are responsible for regulating the size of the pore’s opening. By adjusting the size of the opening, the guard cells control the rate of CO₂ and O₂ uptake and the loss of water by the leaf. In this way, by regulating the diffusion of CO₂ into the cells, the guard cells also control the rate of photosynthesis in the leaf. The guard cells swell when they are full of water, opening the stoma into air spaces that surround the middle layer of leaf cells. This middle layer of cells is called the mesophyll (meso=middle, phyll=leaf). The mesophyll cells are covered with a thin layer of water from the xylem. The water coating the cells evaporates due to the lower water potential in the outside air. New water molecules then move onto the mesophyll cells by osmosis from the xylem. As each water molecule moves onto a mesophyll cell, it exerts a pull on the column of water molecules in the xylem, from the leaves to the roots (see Figure 3). This transpirational pull is caused by the cohesion of water molecules to one another due to hydrogen bond formation, and by the adhesion of water molecules to the walls of the xylem cells. The upward transpirational pull on the fluid in the xylem causes negative pressure to form in the xylem, pulling the xylem walls inward and creating decreased water potential inside the xylem. This decrease in water potential, transmitted all the way from the leaves to the roots, causes water to move inward from the soil, through the root hairs and into the xylem. If the moisture content in the mesophyll layer of the leaf equals or is less than the moisture level of the outside air, the guard cells will lose their water, and the cells will become flaccid and close.

Many environmental conditions influence the opening and closing of the stomata and thus affect the rate of both transpiration and photosynthesis. Temperature, light intensity, air currents, humidity and the nature of the plant all influence the guard cells to open or close.

Experiment Overview

In this laboratory, the rate of transpiration will be measured under various laboratory conditions using a potometer. A potometer is a device that measures the rate at which a plant draws up water.

Materials

Safety Precautions

Scalpels are sharp instruments; use caution when cutting, always cut away from your body and away from others. Although the materials in this lab activity are nonhazardous, follow normal safety precautions. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

1. Place two test tube clamps on opposite sides of the support stand (see Figure 4).
   {10797_Procedure_Figure_4}
2. Gently place the tapered tip of the 0.5-mL pipet into the hole of the #3 stopper. Slide the stopper up until it is at about the 0.25-mL mark.
3. Place the tapered tip of the 0.5-mL pipet into the piece of clear plastic tubing.
4. Wrap electrical tape around the clear plastic tubing 2" from end. Continue until large enough for test tube clamp to hold securely. Approximately 90 cm of tape is needed.
5. Place the plant into a small plastic bag, leaving the stem sticking outside the bag (see Figure 5).
   {10797_Procedure_Figure_5}
6. Complete the next three steps quickly.
   1. Use a scalpel to cut the plant stem from the roots just above the surface of the soil.
   2. Wrap a rubber band around the bag, creating a moderately tight seal to keep water off of the leaves of the plant in steps 7 through 10. The rubber band will be used later to create a seal around the plant stem.
   3. Place the plant stem into the pan of water.
7. Submerge the tubing and the pipet in a pan of water. Use the syringe to draw water through the tubing until all the air bubbles are eliminated. Note: Air bubbles will stop the water from entering the stem of the plant.
8. Use the scalpel to create a new cut on the plant stem while it is under water. Note: This step must be done under water. It is very important that no air bubbles be introduced into the xylem.
9. While the plant and tubing are submerged, insert the freshly cut stem into the open end of the tubing.
10. Have one group member hold the plant leaves out of the water while a second member completes the following steps.
    1. Secure the rubber band around the tubing with the stem inside.
    2. Place a generous amount of petroleum jelly around the top of the tubing and the stem junction to seal the opening. Note: Do not put petroleum jelly on the end of the stem because it will interfere with osmosis.
11. Bend the tubing upward into a “U.” Use the clamps on the support stand to hold both the stopper and taped section of tubing (see Figure 6). Note: Do not allow any air bubbles into the potometer. If an air bubble appears, quickly immerse the pipet, tubing, and part of the stem (but not the leaves) into the pan of water. Use the syringe to flush the air bubble from the potometer. Note: If an air bubble touches the cut end of the stem, then the stem must be cut again under water.
    {10797_Procedure_Figure_6}
12. Remove the bag from the leaves and let the potometer equilibrate for 10 minutes.
13. Expose the plant in the tubing to one of the following assigned treatments. Record the proper treatment condition on the Transpiration Worksheet.
    1. Room conditions.
    2. Gentle breeze—Place a fan at least 1 meter from the plant, on low speed.
    3. High humidity—Mist leaves with water and cover with a transparent plastic bag leaving the bottom of the bag open.
    4. Strong light—Floodlight with heat sink or overhead projector light.
14. After 10 minutes, read the level of water in the pipet. Record the value as time zero in Table 1 on the Transpiration Worksheet.
15. Continue to record the water level in the pipet every 3 minutes for 30 minutes, recording the data in Table 1 on the Transpiration Worksheet. Note: This is the water loss of the plant through the leaves due to transpiration.
16. Calculate the total surface area of the leaves.
    1. Use scissors to cut all the leaves off the plant and blot them dry. Mass them to the nearest thousandth of a gram (0.001 g) on a balance. Record the mass in grams (g) on the Transpiration Worksheet.
    2. Use a ruler and scalpel to cut a 1-cm² section out of one leaf.
    3. Mass the 1 cm² section to the nearest thousandth of a gram (0.001 g) on the balance. Record the mass in grams (g) on the Transpiration Worksheet.
    4. Calculate the mass per square meter of leaf by multiplying the 1-cm² section’s mass by 10,000. Record the result (g/m²) on the Transpiration Worksheet.
       {10797_Procedure_Equation_1}
    5. Calculate the leaf surface area by dividing the total mass of the leaves by the mass per square meter. Record the value (m²) on the Transpiration Worksheet.
       {10797_Procedure_Equation_2}
17. Calculate the water loss per square meter of leaf surface by dividing the water level (loss) at each reading in Table 1 by the leaf surface area calculated in 16e. Record the results in Table 2 on the Transpiration Worksheet.
18. Record the results on the class overhead.
19. Record the averages of the class data for each treatment in Table 3 on the Transpiration Worksheet.
20. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10797_Student1.pdf