This article reprinted from: Pardini, E. A Transpiration: An inquiry-based adaptation of a traditional cookbook lab.

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1 This article reprinted from: Pardini, E. A Transpiration: An inquiry-based adaptation of a traditional cookbook lab. Pages , in Tested Studies for Laboratory Teaching, Volume 29 (K.L. Clase, Editor). Proceedings of the 29th Workshop/Conference of the Association for Biology Laboratory Education (ABLE), 433 pages. Compilation copyright 2008 by the Association for Biology Laboratory Education (ABLE) ISBN All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright owner. Use solely at one s own institution with no intent for profit is excluded from the preceding copyright restriction, unless otherwise noted on the copyright notice of the individual chapter in this volume. Proper credit to this publication must be included in your laboratory outline for each use; a sample citation is given above. Upon obtaining permission or with the sole use at one s own institution exclusion, ABLE strongly encourages individuals to use the exercises in this proceedings volume in their teaching program. Although the laboratory exercises in this proceedings volume have been tested and due consideration has been given to safety, individuals performing these exercises must assume all responsibilities for risk. The Association for Biology Laboratory Education (ABLE) disclaims any liability with regards to safety in connection with the use of the exercises in this volume. The focus of ABLE is to improve the undergraduate biology laboratory experience by promoting the development and dissemination of interesting, innovative, and reliable laboratory exercises. Visit ABLE on the Web at:

2 Transpiration: An Inquiry-Based Adaptation of a Traditional Cookbook Lab Eleanor A. Pardini Biology Department Washington University in St. Louis One Brookings Drive, Box 1137 St. Louis, MO epardini@wustl.edu Abstract: Here I present an inquiry-based adaptation of a traditional lab to plant teach transpiration, the process that supplies leaf cells with water and dissolved minerals, that cools leaves, and is an important part of the global water cycle. Students begin lab by observing stomata on both surfaces of leaves and speculate about the patterns they find. They then work in teams to determine how to use potometers to measure the rate of transpiration. They work in groups and follow a lab handout to design and execute an experiment to test the effect of environmental variables on the rate of transpiration.

3 Poster Session 407 Introduction Transpiration is the process by which water moves through plants from roots to small pores on leaf surfaces, where the water then evaporates as vapor into the atmosphere. Transpiration supplies leaf mesophyll cells with the water needed for photosynthesis, cools leaves, and delivers dissolved minerals from the roots for biosynthesis within the leaf. Only about 1% of the water transported from the roots to the leaves of a plant is actually used for photosynthesis leaving an enormous amount of water that is lost through stomata as it evaporates as water vapor, making transpiration an important part of the global water system. Transpiration is typically taught in a cookbook-style lab in which students follow instructions to carry out a pre-designed investigation. Many versions of this traditional lab for Intro and AP Biology are easily found with an Internet search. In this inquiry-based adaptation of the traditional lab format, students figure out how to use the potometers on their lab benches and then design their own experiments. This lab emphasizes the process of scientific inquiry at the same time it demonstrates an important organismal process in action. Instructor s Notes Materials Potometers One ring-stand with a clamp (for plant) One ring-stand with a ring (for funnel) Rubber tubing and 2 tubing clamps One T or Y-shaped tubing connector Plastic funnel and clothespin (to secure the funnel to the funnel-ring) Plastic pipette with 1/100 ml gradations Plant cuttings (wax myrtle, honeysuckle, and other shrubs work well) Beaker or tray to catch water overflow from the pipette Bucket or tub of water to store plant cuttings and make fresh cuts under water Environmental variables Utility or similar light bulbs (~heat, light) Fans (~wind) Mist sprayers and plastic bags (~to create and trap humidity) Hair dryers (~wind or warm wind) General Lab Outline Prior to lab, assemble the potometers on the lab benches. Set out equipment to measure environmental variables on the lab benches; students will choose what they want to measure and how. During lab, move around the classroom to facilitate group discussion and critical thinking as students figure out how to use the potometers. Encourage groups that figure something out to share their discoveries with classmates until all groups can use the potometers to measure transpiration. Then continue to facilitate discussion as students design their experiments, develop their protocols

4 408 ABLE 2007 Proceedings Vol. 29 and methods, and design tables to record data. Refer to the poster for details on challenges included in the lab handout. Potometer assembly Potometers can be assembled with two ring stands as shown in Figure 1 or with all clamps attached to a single ring stand. Assemble potometers in the morning before the first lab of the day starts. To get started, place a tub of water nearby and under water, make a fresh sloping cut on a branch stem, and keep the stem under water. Turning to the photometer assembly, mount the funnel on the ring stand and attach the three pieces of rubber tubing (A, B, C), plastic T-shaped connector, and pipette (see Figure 1). Apply one clamp to the B tube and then fill the funnel with water. 1. To attach the plant branch, you will need water to flow between the funnel and the rubber tubing leading to the branch. Cut off water flow to the pipette by placing a clamp on the C tube. Remove the clamp from the B tube to allow water to fill the A tube (allow it to overflow and then pinch it off). Quickly insert the recently cut branch stem into the A tube and make sure there are no air bubbles in the tubing. Firmly attach the branch to the ring stand with a clamp (see Figure 1). 2. To set the potometer to the zero point, unclamp water flow to the pipette (tube C) to allow water to flow through the pipette (catch the overflow in the dishpan or sink). Make sure the water in the funnel does not run out as it will introduce air bubbles into the system you can refill it with water from a beaker. 3. To start the transpiration run, clamp off the B tube between the T connector and the funnel so that water can only flow between the pipette and branch stem. Figure 1. Potometer assembly with two ringstands, one clamp to hold the plant branch, and one clamp for a funnel holder. A plastic funnel, the plant branch, and the pipette are connected via rubber tubing with a plastic T-shaped tubing connector (three pieces of rubber tubing are marked A, B, C). Tubing clamps are placed on tubes A, B, and C to control water flow to start and stop measuring transpiration. The funnel is used to allow water through the pipette to reset the water to the zero mark. A tray can be used to catch water overflow from the pipette. Air bubbles in the potometer systems will cause problems as will embolisms in the xylem of the branches. If a potometer is not working well, try recutting the stems underwater and putting the system back together keeping the cut stem underwater as long as possible. After inserting the plant branch, petroleum jelly can be applied to the tubing around the branch stem to prevent water leakage. Students should record increased transpiration rates with the light and wind, but may get low transpiration rates instead if the plants are abused by too much heat or wind. The plants should be kept in dim light between labs so that the baseline (normal room conditions) rate is lower than the

5 Poster Session 409 rate with the spotlight. If plants are used on multiple days throughout the week, leave the plant stem open to the funnel overnight and between labs so it can have a water supply. Comparing rates of transpiration Use instructor-led discussion to guide students to figure out how to compare rates of transpiration among branches. They should arrive at a method that basically allows them standardize plants for the number of stomata/leaf surface area. You can choose to have them actually carry out the comparison (and compare rates among groups) based on the time available and the number of cut plant branches available (sampling leaves is destructive so the number of plant branches you have may dictate if branches need to be used for multiple lab sections). The rate of transpiration is measured as the amount of water lost/m 2 / minute. Because water evaporates through the many stomata on the leaf surface, the rate of transpiration is directly related to the surface area. To arrive at the rate of transpiration, therefore, you must calculate the leaf surface area of each plant: Cut of all the leaves on the plant branch. Calculate the total surface area of all the leaves using the leaf trace or leaf mass method. 1) Leaf Trace Method: Arrange all the cut-off leaves on a 1 cm grid and then trace the edge pattern directly on to the grid. Count all of the grid cells that are completely within the tracing and estimate the number of grids that lie partially within the tracing to sum the total leaf area. 2) Leaf Mass Method: Cut a 1 cm 2 section of one leaf and mass the section. Multiply the section's mass by 10,000 to calculate the mass per square meter of the leaf. (g/m 2 ). Mass all the leaves and then divide the total mass of the leaves by the mass per square meter. This value is the leaf surface area. To calculate the water loss per square meter of leaf surface, divide the water loss at each time interval by the leaf surface area you calculated. Acknowledgements This lab was adapted by E. A. Pardini and R. Specker between for Introduction to Plant Biology Lab (PBIO 1210L) in Plant Biology at the University of Georgia and was based on a lab originally developed by M. Darley and R. Specker. L. B. Brouillette, K. Kulkarni, and W. L. Li provided useful feedback on its use in their lab sections. About the Author Eleanor Pardini is a postdoctoral research associate at Washington University in St. Louis. She earned her Ph.D. in Plant Biology from the University of Georgia in While at Georgia she served as a teaching assistant and lab coordinator in Plant Biology where she taught plant taxonomy and helped to revise the curriculum and lab manual for introductory plant biology labs. She was also an instructor for a learning in biology seminar offered through Academic Enhancement. She served as a university-wide TA Mentor and received the Outstanding TA and Excellence in Teaching awards at University of Georgia Eleanor Pardini

6 Transpiration: An inquiry-based adaptation of a traditional cookbook lab Eleanor A. Pardini, Washington University in St. Louis, St. Louis, MO and University of Georgia, Athens, GA INTRODUCTION Transpiration is the process by which water moves through plants from roots to small pores on leaf surfaces, where the water then evaporates as vapor into the atmosphere. Transpiration supplies leaf mesophyll cells with the water needed for photosynthesis, cools leaves, and delivers dissolved minerals from the roots for biosynthesis within the leaf. Only about 1% of the water transported from the roots to the leaves of a plant is actually used for photosynthesis leaving an enormous amount of water that is lost through stomata as it evaporates as water vapor, making transpiration an important part of the global water system. Transpiration is typically taught in a cookbook-style lab in which students follow instructions to carry out a pre-designed investigation. Many versions of this traditional lab for Intro and AP Biology are easily found with an Internet search. In this inquiry-based adaptation of the traditional lab format, students figure out how to use the potometers on their lab benches and then design their own experiments. This lab emphasizes the process of scientific inquiry at the same time it demonstrates an important organismal process in action. COMPARISON BETWEEN LABS Traditional approach Instruction driven Students follow a list of instructions to set up potometers Students follow a list of instructions and are assigned an experimental treatment Students fill in data tables and graphs provided in lab handouts Students follow formulas to compare transpiration rates among plants Potometers MATERIALS One ring-stand with a clamp (for plant) One ring-stand with a ring (for funnel) Rubber tubing and 2 tubing clamps One T or Y-shaped tubing connector Plastic funnel and clothespin Plastic pipette with 1/100 ml gradations Plant cuttings Left panel: Potometer with one ring-stand holding two clamps (for plant and funnel) and Y-shaped tubing connector. A clothespin is used to clamp the funnel to the funnel-ring and a rubber stopper with hole can be used to stabilize the plant. Middle panel: Potometer with two ring-stands, one with a clamp for the plant, and one with a clamp for the funnel, holder, and a T-shaped tubing connector. In both setups, the funnel is used to allow water through the pipette to reset the water to the zero mark. A tray can be used to catch water overflow from the pipette. Tubing clamps are used to control water flow. Inquiry-based approach Student driven Guided by the instructor, students figure out how to use potometers Guided through the process of inquiry, students design their own experiment and choose their treatments Students design tables and graphs for data collection and presentation Students design a method to compare transpiration rates among plants Environmental treatments Utility or similar light bulbs Fans Mist sprayers Plastic bags Hair dryers Methods TRADITIONAL APPROACH Place the tip of a 0.1 ml pipette into a 16 -inch piece of clear plastic tubing. 1. Submerge the tubing and the pipette in a shallow tray of water. Draw water through the tubing until all the air bubbles are eliminated. 2. Carefully cut your plant stem under water. This step is very important because air bubbles must not be introduced into the xylem. 3. While your plant and tubing are submerged, insert the freshly cut stem into the open end of the tubing. 4. Bend the tubing upward into a "U" and use the clamp on a ring stand to hold both the pipette and the tubing. 5. If necessary use petroleum jelly to make an airtight seal surrounding the stem after it has been inserted into the tube. Do not put petroleum jelly on the end of the stem. 6. Let the potometer equilibrate for 10 minutes before the time zero reading. 7. Expose the plant in the tubing to one of the following treatments (you will be assigned a treatment by your teacher): 1.Room conditions. 2.Floodlight (over head projector light). 3.Fan ( place at least 1 meter from the plant, on low speed). 4.Mist ( mist leaves with water and cover with a transparent plastic bag; leave the bottom of the bag open). 8. Read the level of water in the pipette at the beginning of your experiment (time zero) and record your finding in Table Continue to record the water level in the pipette every 3 minutes for 30 minutes and record the data in Table At the end of your experiment, cut the leaves off the plant and mass them. Remember to blot off all excess water before massing. Analysis of Results 1. Calculate the average rate of water loss per minute for each of the treatments. 2. Explain why each of the conditions causes an increase or decrease in transpiration compared to the control. 3. How did each condition affect the gradient of water potential from stem to leaf in the experimental plant? 4. What is the advantage to a plant of closed stomata when water is in short supply? What are the disadvantages? 5. Describe several adaptations that enable plants to reduce water loss from their leaves. Include both structural and physiological adaptations. 6. Why did you need to calculate leaf surface area in tabulating your results? INQUIRY-BASED APPROACH Challenge 1: Determine how to use a potometer Working in teams, study the potometers on the lab bench and determine how to use such an apparatus to measure the rate of transpiration of a leaf or plant. Which direction do you expect the water in the pipette to move during leaf transpiration? Why? What would happen to the water in the pipette if the rate of transpiration were to increase? Why? What are some environmental variables that can influence the rate of transpiration? How do you think they would affect transpiration? How can you simulate some of these environmental variables in the lab? Challenge 2: Design an experiment to examine the effect of environmental variables on the rate of transpiration Working as a lab team, design an experiment to test the effect of an environmental variable on the rate of transpiration in your plant. You can run your experiment once in ambient room conditions and, after waiting 5-10 minutes, you can repeat your experiment and alter the environmental variable. Address the following issues in your written experimental design: What kind of measurements will you take to record transpiration? What units will you use to report the rate of transpiration? What will your data table look like? What are the column headings? Design a method to compare the rates of transpiration between two different plants. Question: <space to fill in> Hypothesis: <space to fill in> Prediction: <space to fill in> Methods: <space to fill in> Challenge 3: Measure the rate of transpiration Carry out the experiment you have designed and record the data in your data table. Graph your results on graph paper and attach it to your lab activity. Make sure to include titles, axis labels, and units for tables and figures. Table 1. <space to fill in title>. <space to design and record data> Figure 1. <space to fill in title>. Challenge 4: Reflection and discussion 1.Why is transpiration a mixed blessing? 2.Transpiration can be described as solar powered. Why? 3.Does water always move from the roots to the shoots? Why? ACKNOWLEDGEMENTS This lab was adapted for Introduction to Plant Biology Lab in the Plant Biology Department of the University of Georgia by E. A. Pardini and R. Specker, based on a lab developed by R. Specker and M. Darley. L. B. Brouillette, K. Kulkarni, and W. L. Li provided useful feedback on its use in their lab sections.