Measurement of Rates of Aerobic Respiration and Photosynthesis in Terrestrial Plant Leaves Using Oxygen Sensors and Data Loggers - PDF

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Tested Studies for Laboratory Teaching Proceedings of the Association for Biology Laboratory Education Vol. 34, , 2013 Measurement of Rates of Aerobic Respiration and Photosynthesis in Terrestrial
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Tested Studies for Laboratory Teaching Proceedings of the Association for Biology Laboratory Education Vol. 34, , 2013 Measurement of Rates of Aerobic Respiration and Photosynthesis in Terrestrial Plant Leaves Using Oxygen Sensors and Data Loggers William V. Glider and Peter Thew University of Nebraska-Lincoln, School of Biological Sciences, Lincoln NE USA The relationship between aerobic respiration and photosynthesis in plants is a basic biological concept which is often difficult for students to master. This exercise uses terrestrial plant leaves to measure the rate of photosynthesis at different light intensities and wavelengths as well as the rate of aerobic respiration rates of the leaves in the dark. This makes it possible to calculate gross and net photosynthesis and the light compensation point. The exercise employs the Vernier oxygen sensor, Lab Pro data logger, and Logger Pro 3 software linked to a computer for data collection and analysis. Firstpage7 Keywords: plant, respiration, aerobic respiration, photosynthesis, oxygen sensor, leaves, sage leaves Page 1 Spacer Introduction The relationship between aerobic respiration and photosynthesis in plants (specifically angiosperms) is a concept which is often difficult for students to master. This exercise uses terrestrial plant leaves to measure the (1) net rate of photosynthesis at different light intensities and wavelengths and (2) rate of aerobic respiration in the dark. These data are then used to calculate gross photosynthetic rates and the light compensation point. Traditionally, rates of photosynthesis and aerobic respiration in angiosperm leaves have involved a variety of indirect methods including counting the number of bubbles (assumed to be oxygen) released from the stems of the aquatic plant Elodea (Morholt, et.al. 1972), measuring changes in the amount of oxygen in the intracellular spaces of leaf disks in the light and dark (Pitkin 2004), or by measuring changes in carbon dioxide concentration associated with respiration and photosynthesis in Elodea using bromthymol blue as an indicator for the presence of carbon dioxide (Ecklund and Flerlage 2008). More sophisticated equipment for precise measurement of photosynthetic rates can be purchased from Qubit Systems Inc. (Qubit 2013). However, this apparatus is far more expensive than the Vernier oxygen sensors. This exercise employs the Vernier oxygen sensor, Lab Pro data logger, and Logger Pro 3 software linked to a computer to directly quantify the net rate of oxygen evolution by a plant leaf in the light and oxygen consumption by the leaf in the dark. The lab focuses on hypothesis formulation, data collection, and data analysis. The exercise introduces the use of the slope of lines as a measure of rate, and provides the opportunity to discuss the difficulty in measuring photosynthetic rates in living leaves. This lab exercise employs a directed investigative approach and has been used in both majors and mixed majors/ non-majors, large enrollment, introductory biology courses. As written, it can be completed in a three hour lab session but can easily be extended to two lab sessions by using a more investigative approach in which students can test a variety of hypotheses related to other factors affecting rates of photosynthesis and respiration as well as the inclusion of a short exercise involving pigment separation and analysis by William V. Glider and Peter Thew Introduction Major Workshop: Rates of Respiration and Photosynthesis in Terrestrial Leaves Using Oxygen Sensors Student Outline Certain organisms have the capacity to trap light energy directly, and utilize this energy to synthesize high energy monosaccharide sugars from low energy inorganic compounds such as carbon dioxide and water. These simple sugars can then be converted into all other organic nutrients (e.g. complex carbohydrates, lipids, proteins, etc.) required by the organisms to carry out their life processes. These organic compounds can then be oxidized within the cells of these organisms to produce ATP during the process of cellular respiration (aerobic or anaerobic) or used as structural components of cells. Such organisms are classified as photoautotrophs and the biochemical process by which they produce the simple sugars is called photosynthesis. Photoautotrophs can be classified into two groups depending on whether or not they produce oxygen gas as a by-product of the photosynthetic reactions. Certain species of photosynthetic bacteria (e.g. purple sulfur bacteria) use special photosynthetic pigments (bacteriochlorophylls) to trap light energy. During the photosynthetic process a substance other than water (often H2S) is oxidized (loses electrons) with no oxygen being produced as a by-product. Cyanobacteria, plants, and most algae (plant-like eukaryotic organisms including the seaweeds traditionally classified in the Kingdom Protista) use the primary photosynthetic pigment chlorophyll a and a variety of accessory pigments to trap light energy. During the photosynthetic process water is oxidized (loses electrons) with the resulting production of oxygen. In plants and algae the reactions of photosynthesis take place in the chloroplasts whereas in the cyanobacteria the reactions take place on the thylakoid membranes in the cytoplasm. The following equation summarizes the process by which energy from light becomes converted to chemical potential energy in the bonds of the monosaccharide glyceraldehyde 3- phosphate (G3P), which is often converted directly into glucose (C 6 H 12 O 6 ): light 12H 2 O + 6CO 2 G 3 P C 6 H 12 O 6 + 6O 2 + 6H 2 O In plants the process of photosynthesis takes place in two successive steps called the Light Dependent Reactions (light reactions) and the Light Independent Reactions (dark reactions). Each of these two major steps is composed of a series of enzyme-catalyzed biochemical reactions. The light-dependent reactions, also referred to as the light reactions, can take place only in the presence of light. During this phase of photosynthesis, light is absorbed by pigment molecules located in the thylakoids of the chloroplasts. This initiates a series of reactions that result in the conversion of some of the light energy to chemical energy. In the process, Water molecules are split apart, producing hydrogen ions, electrons, and oxygen gas. Some of the oxygen is used by the plant for cellular respiration, but most of it is released into the atmosphere Energy storing ATP molecules are created. The hydrogen ions resulting from the splitting of water are transferred to the hydrogen carrier, NADP, resulting in the formation of NADPH. Thus, in the light-dependent reactions the energy from sunlight is used to make ATP and to reduce NADP+ to NADPH. Some of the captured energy is temporarily stored within these two compounds. Both ATP and NADPH are used in the lightindependent reactions. The light independent reactions, often referred to as the dark reactions or the Calvin Cycle, take place in the stroma of the chloroplast regardless of whether or not light is present. In this series of reactions, the reactions in the stroma use the ATP and NADPH produced in the light-dependent reactions to build simple sugars from CO 2. The light independent reactions can proceed only as long as ATP and NADPH are available. Because ATP and NADPH normally are produced only in the light, the light independent reactions normally stop within a few minutes of the onset of darkness. Note that the light dependent reactions drive the light independent reactions, i.e., the products of the light dependent reactions, namely ATP and NADPH, are required for the light independent reactions to proceed (Fig. 1). Photosynthesis vs. Aerobic Respiration in Plants It is important to remember that plants respire continuously twenty-four hours per day (taking in O 2 and releasing CO 2 ), as all aerobic organisms must do in order to obtain energy to carry on their life activities. A part of the oxygen released during photosynthesis is required for normal respiration, and when the plant is not carrying out photosynthesis (i.e., in the dark) it must obtain respiratory oxygen from the environment. The consumption of oxygen in the dark may be used as a measure of respiration. In the light, with both photosynthesis and respiration proceeding simultaneously, the oxygen evolved is the difference between these opposing reactions. Under optimal conditions, the rate of photosynthesis is twenty times greater than the rate of oxygen consumption by respiration. Hence, under such conditions, the net oxygen measured during photosynthesis represents 90% to 95% of the total oxygen produced. Proceedings of the Association for Biology Laboratory Education, Volume 34, Glider and Thew Figure 1. Relationship between Light Dependent and Light Independent Reactions of Photosynthesis General Procedure 1. Each pair of students will carry out the following three experiments: a. Effect of Light Intensity on Net Photosynthetic Rate of a Leaf b. Effect of Light Wavelength on Net Photosynthetic Rate of a Leaf c. Rate of Oxygen Consumption of a Leaf in the Dark 2. This lab will require the use of a data logger (an electronic data collector) connected to a computer and an oxygen sensor (detector) for the collection of data. The oxygen sensor detects changes in the oxygen concentration in a sealed test tube containing a leaf. a. When using the Logger Pro program, only use those commands given in the procedure. Do not randomly click on menu items. If you mistakenly click on a menu item which takes you to a part of the program that is not called for in the lab exercise, notify your instructor so that he/she can help you return to the required part of the program. b. The oxygen sensor must be kept in an upward position at all times. Therefore, when the sensor is not in use it should be stored upright in the container provided. c. When moving the sensor, grasp it by the upper end; not by the cord! Do not pull on the sensor cord. d. It is extremely important that the sensor is not jostled during data collection since it is likely to result in erroneous data being collected. To limit the possibility of the sensor being hit during data collection, work with the apparatus on your lab desk so that it is located close to the computer rather than being stretched out across the desk. 3. Computer set-up prior to running the experiments: a. You will use a software program called Logger Pro to collect, analyze and display your data. b. Double click on the Photosynthesis Icon on the Windows Desktop. c. Figure 2 is a screen capture of the Logger Pro program as it will appear at the end of data collection. 168 Tested Studies for Laboratory Teaching Major Workshop: Rates of Respiration and Photosynthesis in Terrestrial Leaves Using Oxygen Sensors Figure 2. Screen Shot of Logger Pro Set-Up Collect Button: Click on this button to start data collection. Data collection will stop at the end of the 7 minute data collection period. Analyze Button: Use the drop down menu to display the slope (rate of oxygen production or consumption). Data Button: Use the drop down menu to Clear All Data after each data collection. File: Use the drop down menu to save your data to the desktop or flash drive. Ctrl D: Use for selecting length and rate of data collection. Right Mouse Click: Use for setting graph options. Setting Light Intensities 1. Read the instructions in Appendix A describing the operation of the light meter. Consult your instructor if you have questions on its operation. 2. Hold the light meter sensor on the back of the heat sink so that the white plastic photocell is facing the flood light. Make sure the photocell is held against the heat sink while taking the readings. To insure consistent light measurements at each intensity it will be necessary to place the light sensor head in the exact same position on the heat sink. To insure this is the case, trace an outline of the sensor head on the heat sink with a water soluble marker (vis á vis) pen. Each time you make a light measurement place the sensor head in the traced area. It may be necessary to elevate the base of the light source to insure the light beam irradiates the leaf at a 90 degree angle. Styrofoam blocks are provided for this purpose. To insure that the base of the light source remains in a straight line when moved toward or away from the experimental apparatus, draw lines on the plastic template which represent the sides of the lamp base. 3. By moving the light source base to different distances from the heat sink, identify and mark on the template the following light intensities: 800, 1000, 1500 and 3000 and 7000 fc (footcandles). Leaf Preparation 1. Fill the plastic water bath with 23 o C water and place it on the appropriately marked area on the template. 2. Make sure the inside surfaces of the test tube are dry. Proceedings of the Association for Biology Laboratory Education, Volume 34, Glider and Thew 3. Using a graduated plastic pipet, add 2 ml of the saturated sodium bicarbonate solution to the test tube while keeping the sides of the tube dry. Sodium bicarbonate serves as a carbon dioxide source according to the following equation: NaHCO 3 NaOH + CO 2 4. Using a pasteur pipet, completely fill the micro-centrifuge tube (inserted in a clear tubing base) with tap water. Place a small piece of aluminum foil over the top of the micro-centrifuge tube. Using a push-pin, make a small hole in the center of the aluminum foil. 5. Using a sharp razor blade, excise a leaf from the plant at the base of the petiole. If you are using a pre-harvested leaves (e.g. spinach) cut off the tip of the petiole. 6. Insert the petiole through the foil cover and into the micro-centrifuge tube containing water (Fig. 3). 7. Using the long forceps, place the micro-centrifuge tube containing the leaf into the test tube containing the sodium bicarbonate solution. The leaf may have to be curled slightly in order for it to fit inside the tube. Make sure that the underside of the leaf (lighter in color) does not come in contact with the walls of the tube since this is the side where the majority of the gas exchange takes place via the stomata. 8. Insert the oxygen sensor into the opening of the test tube so that it is tightly sealed. To insure there are no air leaks, wrap a piece of parafilm tightly around the junction of the test tube with the sensor. The final set-up is shown in the figure 4. Figure 3. Leaf Set-up Figure 4. Leaf and Oxygen Sensor Set-up Figure 5. Side View of Experimental Set-up 170 Tested Studies for Laboratory Teaching Major Workshop: Rates of Respiration and Photosynthesis in Terrestrial Leaves Using Oxygen Sensors Apparatus assembly using template (see Figure 5) 1. Fill the plastic heat sink to the top with cold water and place it on the template in the space marked heat sink. Note that the heat sink does not touch the water bath. 2. Insert the test tube containing the leaf into the hole in the water bath cover so that about 2 cm of the tube is sticking out of the top of the cover. 3. Place the wooden base of the light source at the 800 fc mark. Your apparatus should now look like Figure 5. Exercise A: Effect of Light Intensity on the Net Photosynthetic Rate of a Leaf Procedure 1. In this experiment you will be measuring the photosynthetic rate of a leaf at the following light intensities: 1000, 1500, 3000, and 7000 fc. 2. Hypothesis Formulation Based on your knowledge of photosynthesis, formulate a hypothesis concerning the effect of light intensity on photosynthetic rate in leaf cells. Write down the hypothesis before starting the laboratory experiments. 3. Pre-Illumination of the leaf at 800 fc To stimulate the light dependent reactions of photosynthesis to produce measurable levels of oxygen, it is necessary to illuminate the leaf at 800 fc before taking photosynthetic rate measurements. a. Place the wooden base of the light source at the 800 fc mark on the template and make sure the face of the bulb is parallel to the face of the heat sink. Turn on the light for 7 minutes. Do not collect photosynthetic rate data at this light intensity. 4. After equilibrating the leaf at 800 fc for 7 minutes, move the base of the light source to the 1000 fc mark on the template. a. Begin collecting data by clicking on the Collect button on the Tool Bar. The data logger will collect data for 7 minutes. b. As the data is being collected periodically check the following: The current ppm oxygen reading appears in the Meter Window. A graph of the results will appear in the Graph Window. The Y-axis is generally set between 180, ,000 ppm (parts per million) oxygen. However, the initial oxygen reading may lie outside this range. To find where the current ppm oxygen begins, scroll along the Y- axis by clicking on the up or down arrows next to the Y-axis. More precise interval settings for the graph axes can be made by right clicking the mouse and selecting Graph Options from the drop-down menu. 5. After 7 minutes, the data logger will automatically stop collecting data. 6. Obtain the net photosynthetic rate (= slope of the line) for the last 2 minutes of the collection period by doing the following: a. Click the Analyze button on the Menu Bar. b. Select Linear Fit on the drop-down menu. c. A box giving the slope of the entire line will appear on screen. d. Move the first bracket symbol to the 300 sec (5 minutes) mark on the X axis and move the second bracket to the 420 (7 minutes) sec mark. e. Record the slope of the line (net rate of oxygen production or consumption) for the last 2 minutes of the collection period in Table 1a for 1000 fc. 7. Save the data on your flash drive for further analysis. Once the data is cleared from the computer s memory, it can t be restored! Be sure the data has been recorded before starting to collect data at the next light intensity. Proceedings of the Association for Biology Laboratory Education, Volume 34, Glider and Thew 8. Once you have saved your data for 1000 fc, the data must be cleared from memory so that a new trial can be run. Click on the Data button in the menu and select the Clear All Data option at the bottom of the drop down menu. 9. You will now repeat the above procedure for light intensities 1500, 3000 and 7000 fc. Do not remove the oxygen sensor from the tube and do not move the water bath or heat sink. 10. At each light intensity record the slope of the line (= net rate of O 2 evolution or consumption) in Tables 1b 1d. Exercise B: Effect of Light Wavelength on Net Photosynthetic Rate of a Leaf Introduction What is Light? Light is a type of electromagnetic energy (radiation). Electromagnetic radiation travels in waves or packets of light called photons. The distance between the crests of electromagnetic waves is termed the wavelength and is measured in nanometers. The entire range of radiation is known as the electromagnetic spectrum. Each type of radiation in this spectrum has a characteristic wavelength and energy content. These two characteristics are inversely related; i.e. the longer the wavelength, the smaller the energy content. The portion of the spectrum which is detected as various colors by the human eye is referred to as visible light and ranges from about nanometers. White light is composed of all wavelengths (colors) in the visible light spectrum. We see colors because objects contain pigments that selectively absorb certain wavelengths of visible light and reflect or transmit others. What we recognize as an object s color is composed only of those wavelengths of light that are transmitted or reflected. If a pigment absorbs all wavelengths, it appears black. The diagram below illustrates what is meant by the terms incident light, reflected light, and absorbed light. Photosynthetic Pigments in Plants Several different photosynthetic pigments, i.e. those pigments which are able to capture various wavel
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