Laboratory. The Effect of Light Color on Photosynthesis

Transcription

1 Laboratory 7 The Effect of Light Color on Photosynthesis

2 Biology 171L FA17 Lab 7: Effect of Light Color on Photosynthesis Student Learning Outcomes 1. Use an O 2 Gas Sensor to measure the amount of oxygen gas consumed or produced by a plant during respiration and photosynthesis. 2. Determine the rate of photosynthesis of a plant, as measured by oxygen production. Relevant Readings Campbell Biology, Chapter 10, especially pp A Short Guide to Writing about Biology, Chapter 9 Homework Synopsis (see pages 7-9 & 7-10 for full description) Part I Mastering Biology Part II Science Communication First draft of photosynthesis report Part III Data Analysis Grading exercise INTRODUCTION Life on earth could not exist without photosynthesis. Photosynthesis enables plants, algae, and some bacteria (autotrophs) to harness the energy in light and convert it to a usable form of energy. It is the only process that is able to convert light energy to chemical energy. That energy can then be used to create the macromolecules that serve as fuel for cellular respiration in all other organisms (heterotrophs). Photosynthesis can only happen in the presence of light energy and chlorophyll. We often think of sunlight as a single white wavelength, but in reality it is a continuum of wavelengths - each wavelength representing a different color of light. (Figure 1). Light travels in waves and these waves vary in wavelength, which is defined as the distance from the top of one wave to the top of the next wave. Shorter wavelengths, like X-rays, contain more energy, Figure 1. The visible light spectrum is a small portion of the electromagnetic spectrum. Energy is inversely proportional to wavelength, so that long wavelengths, such as radio waves carry less energy than short wavelengths such as gamma rays. Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-2

3 while longer wavelengths, like radio waves, contain less energy. Photosynthetic organisms contain special pigments that absorb light energy for use in photosynthesis. These special pigments are stored in oval-shaped organelles called chloroplasts. Chloroplasts are numerous; between 20 and 40 can be found moving around within a single cell of a green plant. Pigments absorb light energy in a narrow band we know as the visible light range ( nm). The human eye sees the wavelengths being reflected by the pigment. A pigment that appears red to the human eye does so because it is absorbing blue and green light and reflecting orange and red. There are many different pigments and each is distinctive in the wavelengths it absorbs and reflects (Figure 2). The three major classes of photosynthetic pigments are: chlorophylls, carotenoids, and phycobilins. Absorbance of light by chloroplast pigments Wavelength of light (nm) Figure 2. Absorbance spectra of chlorophyll a, chlorophyll b, and the carotenoids. Chlorophyll molecules have a polar head (a porphyrin head) and a non-polar tail (Figure 3). The presence of the non-polar tail helps the chlorophyll molecule embed into the thylakoid membrane. There are multiple chlorophyll pigments. Chlorophylls a and b are utilized by higher plants and green algae. Diatoms and brown algae possess chlorophylls a and c, and red algae uses chlorophyll a and phycobilins. Chlorophyll absorbs light in the red and violet wavelengths, and uses this energy for photosynthesis. Chlorophyll absorbs very little of the green wavelengths, which it reflects. Consequently, green is the color generally associated with plants. The carotenoids (Figure 4) form a family of pigments that absorb light in the blue range giving this class of pigments color which ranges from pale yellow to orange to red. Carotenoids are categorized into two classes, xanthophylls, which contain oxygen atoms, and carotenes, which are mostly hydrocarbon. Carotenoids are the dominant pigments in fruits and flowers giving them their bright vivid colors. They are also present in leaves of plants, but are masked by the chlorophyll present in mature leaves. The colors of carotenoids are apparent in young leaves, ripening fruit, and dying plants, where the levels of chlorophyll have dropped off. Carotenoids Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-3

4 are important accessory pigments. They are also important in photo-protection, that is, they protect plants from harmful UV radiation. Figure 3. Structure of chlorophyll a molecule Figure 4. Structure of several carotenoid molecules The last class of photosynthetic pigments, phycobilins, is not present in land plants or green algae. Instead, phycobilins are found in red algae and cyanobacteria. Phycobilins absorb in the green and orange portion of the spectrum and are important for allowing marine and aquatic plants to harvest the wavelengths not filtered out by the water. PHOTOSYNTHESIS AND RESPIRATION Photosynthesis refers to the process in which light energy converts carbon dioxide and water into sugar, oxygen, and other organic compounds. This process is summarized by the following reaction: 6 H 2 O + 6 CO 2 + light energy (sunlight) C 6 H 12 O O 2 Cellular respiration refers to the process of converting the chemical energy of organic molecules into a form immediately usable by organisms. Glucose may be oxidized completely if sufficient oxygen is available, illustrated by the following equation: C 6 H 12 O O 2 6 H 2 O + 6 CO 2 + energy (ATP) The rate of photosynthesis is affected by numerous factors including the amount and the wavelength of light available. In today s experiment, you will examine how photosynthesis is affected by the color (a.k.a. quality or wavelength) of light available to Elodea leaves by passing light through different colored bulbs. If the wavelength available to the plant affects photosynthesis, then we would expect to see a difference in the photosynthetic rates produced by the different colors of light, and therefore a measurable difference in the amount of O 2 produced during photosynthesis. Total or GROSS photosynthesis, is the total amount of carbon fixed or oxygen Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-4

5 released by a plant in the light. However, because plants are always undergoing respiration, even in the dark, we must subtract the value of respiration from the total, or gross, amount of photosynthesis. In order to compare between different plants and different treatments, we would like to compare the amount of photosynthesis that is left over after the costs of respiration are included. Therefore, the GROSS photosynthetic rate is determined from the photosynthetic rate in the light (photosynthesis + respiration) plus the RESPIRATION (absolute value of rate in the dark). To determine this, you will measure the concentration of O 2 present during a closed reaction. First you will measure the amount of O 2 consumed during respiration (in the dark); then you will measure the amount of O 2 produced by photosynthesis (in the light). You will record the rate of O 2 produced or consumed as the slope of O 2 in ppm/s. You will use the absolute values of your slopes to calculate your GROSS photosynthetic rate. GROSS Photosynthetic Rate= O 2 ppm s -1 in LIGHT + O 2 ppm s -1 in DARK You will also collect an absorbance spectrum of the photosynthetic pigments extracted from Elodea and determine if there is a correlation between the rates of photosynthesis under the different colored bulbs, the wavelengths of light that those filters transmit, and the optimum wavelengths of light absorbed by the plant. EXPERIMENT PROCEDURE Overview You will work in groups, as directed by your TA, and each group will take one part of the experiment. You will either measure the respiration/ photosynthesis rate of Elodea under two of five colors of lights (blue, green, orange, red, and white) (Parts A & B); or, you will record the absorbance spectrum for Elodea (Parts C & D), and measure the respiration/ photosynthesis rate of Elodea under one of five colors of lights (blue, green, orange, red, and white) (Parts A & B). Each color of light (blue, green, orange, red, and white) represents one treatment. You will share your results with the other member in your class to form an initial data set. Additionally, your TA will collect your data and submit it to the Lab Coordinator to be pooled with data from other sections. You will analyze the entire class dataset, giving you approximately 4-6 samples per treatment. Your goal is to determine which color of light is the best driver of photosynthesis. (Hint: Remember that there are two photosystems - PSII and PSI - and the wavelengths they utilize slightly different. How do electron transport chains work?). Part A: Respiration Rate 1. Turn on the computer. Connect the Vernier Lab Pro interface to the computer. If the Lab Pro power cord is not plugged in do so now. The Lab Pro will beep when the power is connected. Connect the O 2 Gas Sensor to the Lab Pro interface. 2. Prepare the computer for data collection. Open Logger Pro 3 by clicking on the Logger Pro icon in the computer desktop dock. Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-5

6 3. Go to File in the menu and click on Open. 4. The Logger Pro window for measuring O 2 production will open. Under Experiment>Data Collection set the Duration to 15 minutes and set the Sampling Rate to 20 samples/minute. Make sure that units are in ppm (go to Experiment>Change Units, if necessary). 5. Find the respiration chamber and wrap the entire respiration chamber in aluminum foil so that no light can enter. 6. Obtain a piece of Elodea from one of the aquaria at the back of the room. Measure and cut a piece 10 cm long. Place the remaining tissue back into the aquarium. 7. Carefully place the Elodea into the respiration chamber. Try to spread the plant out as much as possible in the chamber. 8. Move the lamp so that the bottom of the bulb or the rim of the shade, whichever is lowest, is 7 cm from the table. Do not let the lamp touch the respiration chamber. Make sure that the light is as close to 90 to the respiration chamber as possible. Place the O 2 Gas Sensor into the bottle. Place the sensor/chamber on its side under the fluorescent light on the table. Even though this is the dark control, the light should be turned on (why?). Wait 5 minutes for the chamber to acclimate before proceeding. 9. To begin measuring the O 2 concentration in the chamber, click the green button in the Logger Pro menu bar. Data will be collected for 15 minutes. 10. When data collection has finished, you will determine the rate of respiration. a. Autoscale the data by clicking the Autoscale button on the toolbar. b. Move the cursor to the point where the data values begin to increase. Hold down the left mouse button and drag the cursor to select the region of decreasing oxygen gas concentration. c. Click on the Linear Fit button,, to perform a linear regression. A box will appear with the formula for a best fit line. d. Record the slope of the line, m, as the rate of respiration in Table 1. e. Close the linear regression box. 11. Store your data by choosing Store Latest Run from the Experiment menu. 12. Remove the aluminum foil from around the respiration chamber. Do NOT remove the O 2 sensor from the respiration chamber. Part B: Photosynthesis/Respiration (Net) Rate under Colored Light 13. Position the chamber under the light. Do NOT remove the O 2 sensor from the respiration chamber. 14. If necessary, reposition the lamp so that the bottom of the bulb or the rim of the shade, whichever is lowest, is 7 cm from the table. Do not let the lamp touch the respiration chamber. Wait 5 minutes prior to beginning data collection. 15. After the five-minute acclimation period is up, begin data collection by repeating steps 9- Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-6

7 11 above. When you click on the green Collect button, you will be given a set of options, be sure to choose Store Latest Run. This will allow for all trials to be displayed on the same figure. 16. Remove the Elodea from the respiration chamber. Clean and dry the respiration chamber. Light Color Rate of O 2 production/ consumption in the DARK (ppm/m) Rate of O 2 production/ consumption in the LIGHT (ppm/m) GROSS Rate (ppm/m) (rate in the light plus rate in the dark ) 17. Once you have finished on one side of the table, swap places with your tablemates and perform a second set of measurements. This may be a second color, or the absorbance readings, depending on your table. Part C: Extracting the Photosynthetic Pigments from Elodea 18. Obtain a small amount of Elodea from one of the tanks at the back of the room. Take short pieces that are floating in the tank when ever possible. Blot your piece(s) dry with a paper towel. Weigh out approximately 4.0g±0.2g of Elodea tissue and place it in a 250 ml glass beaker. 19. Cover the Elodea with 95% ethanol (EtOH) (approximately 50 ml). 20. Place the beaker on a hotplate with a stir bar. Adjust the stirrer until the stir bar is just spinning. Heat until the Elodea-EtOH solution is just boiling (approximately 10 minutes). The photosynthetic pigments will be extracted out of the leaves into the EtOH. Remove from the hotplate. 21. Fold a piece of filter paper in quarters, and place it into a funnel. Moisten the filter paper with a few drops of EtOH. 22. Put the end of the funnel into a clean beaker. Pour the pigment extract through the filter paper into the beaker. 23. Take 0.5 ml of the filtered pigment extract and put it into a 50 ml beaker containing 5.0 ml of 95% EtOH 24. Pipette 3.0 ml of the diluted extract into a cuvette for your sample. Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-7

8 25. Pipette 3.0 ml of 95% EtOH into another cuvette for you blank. Part D: Determining the Absorbance Spectrum of Elodea Photosynthetic Pigments Your SpectroVis Plus Spectrophotometer needs to warm up for at least 5 minutes. If it is not on, turn it on now. 25. Calibrate the spectrophotometer using the instructions listed on the page entitled Vernier SpectroVis Plus Spectrophotometer. 26. Insert the cuvette containing your chlorophyll extract. In 20 nm increments, record all absorbance values between 380 nm and 720 nm. 27. When you are done with your readings dispose of the EtOH and the pigment extract in the waste ethanol container. **Entering Data Please note that you will enter data for each light color and the absorbance data onto separate TABS of a Google doc. Similarly, the data will be sent to you in an excel spreadsheet that is set up the same way. You are only required to enter the values you observed in the dark and in the light the spreadsheet will calculate the Gross Photosynthetic Rate automatically. Ensure that your data are captured in the correct units. If you make a mistake, make note of it on the spreadsheet. Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-8

9 Lab 7 Homework Due Week of October 16, 2017 Part 1 Mastering Biology (44 points): A. Answer the questions in the assignment entitled 08. DNA on the Mastering Biology site. You have until the night before lab at 11:59 PM to complete these questions. Part 2 Science Communication (20 points) A. Writing a Full Lab Report (20 points) Using Pechenik, 2013 (A Short Guide to Writing about Biology, Chapter 9, and Gillen, 2007 (Reading Primary Literature) as guides, write a full lab report for this experiment. You must include the following sections: Introduction, Materials & Methods, Results, Discussion, and Reference List. You must turn in two copies of this draft!! Put your secret number on both copies. Find at least one article from the primary literature to use in your Introduction to introduce the experiment, and at least one article to use in your Discussion to support one of your conclusions. Be sure to cite these articles appropriately. Your report must be typed, 12-pt font, Times New Roman font, double-spaced with 1 margins. Follow the guidelines in the Universal Rubric and the Lab Report Grading Template Rubric on Laulima. Your report must consider/ include all the colors of light that were measured, and the absorbance data of the chlorophyll extracted from the Elodea, even if you didn t do the actual experiment. Additionally, you must consider the following questions when you are interpreting your results: 1. Were either the respiration or photosynthetic rate values a POSITIVE number? If so, what is the biological significance of this? 2. Were either the respiration or photosynthetic rate values a NEGATIVE number? If so, what is the biological significance of this? 3. Do you have evidence that cellular respiration occurred in the Elodea? 4. Do you have evidence that photosynthesis occurred in Elodea? 5. Think of at least three factors that might influence the rate of carbon dioxide production or consumption in plants. How do you think each will affect the rate? 6. What is white light? How is it different from red light, or any of the other colors you measured? 7. Why do you think the color of light has an influence on the rates that you measured? 8. How does the absorbance spectrum of the chlorophyll extracted from the Elodea influence which colors are the best drivers of photosynthesis?

10 This paper will be the first of two drafts you will write before you turn in the final paper at the end of semester. In the coming weeks, you will have the opportunity to re-write the report. Your final lab report, which will be due in the last lab, will be graded for content. There are many resources, both on campus and online, to help you succeed. The following websites have many ideas: Your grade this week will be based on how hard you tried, not on the content. In other words, you should put the same amount of effort into this report that you would, if you had only one chance to get it right. For example, if you write the materials and methods section in bulleted form, you will lose points. Or, if you write the introduction, but do not include a journal reference, you will lose points. Criteria Submitted your finished paper on time to allow others to comment on your work Not Completed 0 points Insufficient 5 points Incomplete / not finished/ or past deadline Sufficient 15 points Mostly complete and in on time, e.g., may be missing references to journal articles Expected 20 points Fully complete and in on time Part 3 Data Analysis (5 points) Use the grading template on the following page (and on Laulima) to evaluate the draft you are handing in and give yourself a grade. Attach this template to the copy you will give to your TA. On the back of the template, note the improvements you intend to make in your next draft to improve your lab report and increase your overall grade. Biol 171L - FA17 Effect of Light Color on Photosynthesis 7-10

11 Lab Report Grading Template Name: (3) (2) (1/0) Title YES OK NO Appropriately & concisely describes goals of study (including names of species studied) Title Total /3 Introduction YES OK NO Begins with brief overview of the big picture All background information (with citations) is relevant, leads to statement of intent and any anticipates all aspects of the work performed hypotheses, and All statements are supported with refs or examples Relevant literature summarized & correctly cited Includes clear statement of specific issues to be addressed Introduction Total /15 Methods YES OK NO Begins with general overview of the system & approach Important points of experimental procedures described Includes description of what variables were measured Includes brief description of how data were analyzed Methods Total /12 Results YES OK NO All statements are supported with reference to data Findings complete & structured around research questions posed in the Introduction Figures & tables are self- sufficient & clear Results presented succinctly & not over or under- interpreted Results Total /12 Discussion YES OK NO All statements are supported by refs, data, or example Data are discussed wrt expectations raised in the Introduction Presents alternate hypotheses if results fail to match predictions Discusses valid problems with experimental design Addresses how the present results compare to those of previous studies Paper presents positive, logical suggestions for future work Discussion Total /18 General Format & Writing Style Clearly written /3 Carefully proofread, pages numbered /3 Includes expanded literature cited, in proper format /4 Extras Extra papers read & referenced Data used to address new questions Total Lab Report Grade /70