AP Biology Core Lab Transpiratio

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					AP Biology Core Lab: Transpiration
Introduction: The amount of water needed daily by plants for the 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 leaves). 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 upward in pull as a result of transpiration, which creates a negative tension. This “pull” on the water from transpiration is increased as a result of cohesion and adhesion of water molecules. The details: Transpiration begins with evaporation of water through the 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 that the outside air and water tends to evaporate from the leaf surface (moving from and 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 transported pull is caused by (1) the cohesion of water molecules to one another due to hydrogen bond formation and (2) 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 water 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. In this investigation you will compare transpiration rate in different environmental conditions: Light, humidity and high air currents. You will also make your own cross section of a stem to view under the microscope.

Materials Part A: Transpiration plant cutting ring stand with 2 clamps rubber tubing .1 mL pipette petroleum jelly fan lamp plastic bag with spray bottle of water for misting electronic balance metric ruler

Part B: Stem Structure plant stem nut and bolt microtome razor blade melted paraffin dish of 50% ethanol dish of toluidine blue stain dish of distilled water forceps slide with coverslip compound microscope

Procedure: Note: lab teams are groups of four students. Two students will proceed to the procedure for Part B and the other two will set up Part A. PartA: Transpiration 1. Set up a potometer (see diagram) in the following manner;  Put the rubber tubing and pipette in the clamps on the ring stand with the end of the rubber tubing about half the height of the graduated pipette; see diagram.  Fill the 10 mL syringe with water and then attach it to the T connector of the photometer  Add water from the syringe until the level of water forms a bead on top of the rubber tubing  Cut your plant stem diagonally while under water with a clean razor blade  Quickly place the cut end of the stem in the rubber tubing such that at least a ½” is inserted; making sure that the stem fits snugly.  Create an air-tight seal around the stem with petroleum jelly.

2. Let the potometer equilibrate for several minutes (10) before recording a time zero reading 3. Each lab group will set up ONE of the environmental conditions on the back of this page:

4. Read the level of water in your pipette every 3 minutes for thirty minutes. Record the data in Table I and get data for the other environmental conditions from other teams. 5. Take all of the leaves off of your plant, pat them dry and then mass them. Cut a 1 cm2 square from one leaf and mass this. Multiply the 1 cm2 section by 10,000 to calculate the mass/meter2 of leaf. Then divide the total leaf mass by the mass/meter2. Mass of total leaves ________ Mass of 1 cm2 section ___________ Total Mass of leaves (grams) = Leaf Surface Area (m2) g/m2 Part B: Stem Structure 1. Obtain a nut-and-bolt microtome and turn the nut until it is almost at the end of the bolt forming a small cup. 2. Cut a short piece of plant stem (about 5 mm) slightly longer than the depth of the cup in the nut. Stand the stem on its end in the opening of the nut and carefully pour melted paraffin over it. 3. Let the wax solidify for a few minutes and then hold the head of the bolt horizontal with one hand and slice down holding the razor blade with your other hand. Twist the bolt a little so that a thin core of paraffin and stem sticks up above the surface of the nut. Use a slicing motion to cut this section down to the nut. See diagram below.

4. Cut multiple thin slices and put them in a dish of ethanol. Leave them for five minutes. Free the tissue from the paraffin if necessary and move them with forceps to the dish of toluidine blue stain. Leave them in the stain for 1 – 3 minutes. 5. Rinse the sections in a dish of distilled water and make a wet mount with one slice section, using a drop of glycerin instead of water under the cover slip. View the slide under the compound microscope and determine whether this is a monocot or dicot.

Name _______________________________________________ Date _________________ AP Biology Core Lab: Transpiration and Stem Structure Data Part A: Transpiration

*calculated leaf surface area _________________m2

*to calculate Water Loss (mL), subtract the values in Table 1 (3 – 0, 6 – 9, 12- 9, etc) *to calculate Water Loss per m2, divide Water Loss by Calculated Leaf Surface Area in m2

Make a line graph of the cumulative water loss in mL/m2 of the plants under different environmental conditions. Graph each line in a different color, providing a key. Independent variable _________________________________________________ Dependent variable _________________________________________________

Graph Title: _______________________________________________________

Discussion 1. Calculate the average rate of water loss per minute for each of the treatments by dividing the total water loss by the total number of minutes observed. Show work! Room: ___________________________________________________________ Fan: ______________________________________________________________ Light: _____________________________________________________________ Mist: ______________________________________________________________ 2. Which conditions increased transpiration? _____________________________________________ 3. Discuss why in terms of water potential. _______________________________________________ ________________________________________________________________________________ _________________________________________________________________________________

4. Which condition(s) decreased transpiration and why? ______________________________________ ________________________________________________________________________________ ________________________________________________________________________________ ________________________________________________________________________________

5. Why was it necessary to calculate leaf surface area in this lab? _________________________________________________________________________________ _________________________________________________________________________________ _________________________________________________________________________________

6. Based on stem structure, is your plant a monocot or dicot? Explain. _________________________________________________________________________________ _________________________________________________________________________________