Lab 3: Photosynthesis & Respiration I

BIOL 153L General Biology II Lab Black Hills State University Lab 3: Photosynthesis & Respiration I Photosynthesis is the process by which plants utilize light energy to fix carbon into chemical energy in the form of sugar. The basic formula of photosynthesis is: carbon dioxide + water + light energy» carbohydrate + oxygen + water The light reactions of photosynthesis occur in the chloroplast of photosynthetic eukaryotes. In the chloroplast, photosynthetic pigments, which include green chlorophyll as well as pigments of other colors, absorb light. The energy from the light is used to strip electrons from water, which releases oxygen (O 2 ) as a waste product. (Photosynthetic organisms are in fact responsible for the relatively high levels of oxygen in the Earth's atmosphere!) NADPH and ATP are also generated during the light reactions; these molecules are important later in the process of photosynthesis. True to their name, the light reactions only occur when a photosynthetic organism is exposed to visible light. In the dark reactions of photosynthesis, short-lived but high-energy compounds produced by the light reactions (i.e., NADPH and ATP) are used to fix CO 2 into sugar using a complex biochemical pathway. Carbon dioxide enters cells via stomata (on the leaves of true plants) or via diffusion (for algae and protists). In a multi-stepped process, the carbon dioxide is reduced the carbon is fixed and becomes part of a sugar molecule, and the oxygen combines with hydrogen ions to become water. Note that the dark reactions do not require light, and may occur in a photosynthetic organism in dark or light environments. However, if the photosynthetic organism is deprived of light for too long, it will run out of the energy (ATP and NADPH) needed to run the dark reactions. Like animals and other heterotrophs, plants break down food molecules for energy via respiration. The difference is they are breaking down food (i.e., sugars, complex carbohydrates, fats, proteins) that they made directly or indirectly via photosynthesis. Respiration occurs in the mitochondria, and food molecules (sugars) are oxidized to release energy in the form of ATP and oxygen is consumed by this reaction, while carbon dioxide is released. The basic formula of respiration is: carbohydrate + oxygen + water» ATP (energy) + water + carbon dioxide Note that for photosynthetic organisms, respiration and photosynthesis are opposing biochemical processes plants release carbon dioxide and use oxygen in respiration, but release oxygen and use carbon dioxide in photosynthesis. Ultimately, for plants to grow and successfully reproduce, the carbohydrate gains from photosynthesis must exceed the carbohydrate losses from respiration. Photosynthesis in land plants may be studied using expensive instrumentation, like a leaf porometer, that estimates gas exchange across leaf stomata. In many ways, aquatic environments are better suited for this kind of research. Like their counterparts on land, photosynthetic organisms in aqueous solutions will consume and release carbon dioxide and oxygen based on the balance of respiration and photosynthesis but amounts of these gasses may be easily measured because they remain trapped in the water rather than escaping to the atmosphere. Thus, photosynthetic rates may be measured by ph (solutions with abundant carbon dioxide are acidic, owing to the formation of carbonic acid) or formation of air bubbles (which are oxygen molecules produced by the light reactions of photosynthesis. Today we will use air bubbles and their buoyant properties to look at factors influencing light and dark reactions of photosynthesis. 1

Introduction to "Leaf Disks" Although terrestrial plants normally undergo gas exchange with the atmosphere, their leaves will carry out dark and light reactions of photosynthesis for a day or two following their submersion in water. Thus, we can estimate photosynthetic rates of higher plants in the absence of water limitation by placing leaf pieces in aqueous solutions and measuring concentrations of carbon dioxide (e.g., by determining ph of the water) or oxygen (e.g., by evaluating oxygen bubble formation). The latter is particularly easy to assay, because oxygen bubbles make leaves buoyant the rate of photosynthesis will be linked directly to the time it takes leaf pieces to float in water! Today you will examine factors that affect photosynthesis such as availability of carbon dioxide, amount and wavelength of light energy, and ambient temperature but not water related factors like air humidity and soil moisture by observing leaf disks of ivy (Hedera helix and H. hibernica) in an aqueous bicarbonate solution. You will first use vacuum infiltration to remove oxygen from the leaf disks and replace it with water; this causes the disks to sink. Following this treatment, oxygen that accumulates in intercellular spaces of the leaf spongy mesophyll via photosynthesis will cause disks to rise from the bottom of their container and approach the water surface. The time that it takes for leaves to rise in the aqueous solution is a measure of photosynthetic rate achieved under conditions prescribed to the container (e.g., availability of carbon dioxide, light exposure, temperature, etc.). Anatomic features of plant leaves will be covered in depth later in the BIOL 153 labs. Nonetheless, it is worth reviewing basic leaf structures before proceeding with the exercise today. Stomata are located primarily on the bottom of leaf surfaces in ivy. Inside these stomata are loosely packed cells (spongy mesphyll) with abundant air spaces; this is the primary site of gas exchange and, of significance to today's lab, accumulation of oxygen for leaf disks in an aqueous solution. Above the spongy mesophyll is the palisade mesophyll one or more rows of densely packed cells in which the light and dark reactions of photosynthesis occur and then epidermis. Vascular tissue (xylem and phloem) is contained in leaf veins that transect the spongy and palisade mesophyll. The images below show cross sections of a hypothetical plant leaf with aforementioned structures and layers. 2

Getting Organized: Watch the ksbioteacher video, Sinking Leaf Disks, before proceeding: https://www.youtube.com/watch?v=vw8bazo89oc 1. You will be working together in groups of 3-4. Please move to a new table if you are alone or have a single partner at your table. 2. Check that the following items are available on your lab table. Filled beaker marked Sodium Bicarbonate Solution Filled beaker marked Distilled Water 4 syringes (without needles) 6 small plastic cups 1 paper punch 1 lamp 1 large Petri dish 1-2 probes Ivy leaves Three paper labels indicating treatment 3. Each group member should practice using the syringe to create a vacuum. Use beaker marked Distilled Water for this exercise. Use syringe to draw up 10 ml of distilled water then point syringe toward ceiling and carefully push plunger to expel air bubbles. Once air bubbles are expelled, place a thumb or index finger from one hand firmly over the syringe tip. Use the other hand to pull the plunger. You should feel resistance! Hold for a few seconds and then release. 4. Each group member should practice emptying the syringe contents into a plastic cup without spilling. Pull plunger almost but not all the way out of the syringe, then hold thumb over syringe tip. Hold the syringe over the cup and carefully remove the plunger fully. Dump the water into the plastic cup, and then dispose of this water in the lab sink. Experiment 1: Testing effects of carbon dioxide concentration and light intensity on oxygen production in ivy leaf disks. Let's start today by looking at two basic factors that we'd expect to affect photosynthetic rates the availability of carbon dioxide and light! Motivation and Design of Experiment #1, Part A Observation: Water naturally contains small amounts of CO 2 that photosynthetic organisms use to fix carbon during the dark reaction of photosynthesis. Question: Would photosynthetic rates change if additional carbon is added to the water? Method: Addition of baking soda (sodium bicarbonate, NaHCO 3 ) to water will increase the concentration of carbon dioxide in the solution. Thus, a solution of sodium bicarbonate will be used to infiltrate a set of leaf disks used in the photosynthesis assay. Control: A second set of leaf disks will be infiltrated with distilled water (lacks sodium bicarbonate and other impurities) to compare with above-described NaHCO 3 infiltrated leaf disks ANSWER THE QUESTIONS BELOW BEFORE STARTING EXPERIMENT! 3

1. Why is baking soda added to the water in this experiment? 2. Why will the leaf disks float when photosynthesis occurs? Describe what fast photosynthetic rated will look like relative to a low photosynthetic rate (i.e., how can you tell a difference)? 3. Do you predict that photosynthetic rates will be higher or lower when baking soda is added? Motivation and Design of Experiment #1, Part B Observation: Plants require light to grow. Question: Do the light reactions of photosynthesis occur in leaves placed in the dark? Method: Prevent light exposure from contacting one container of leaf disks that is infiltrated with sodium bicarbonate, and then assay photosynthetic rate on these leaf disks over time. Control: A second set of leaf disks will be exposed with light to compare with the above-described dark-treated container of leaf disks. 4. Why are both leaf disk containers (light + dark treated) infiltrated with sodium bicarbonate? Would it be okay to use distilled water for one leaf set? Explain your logic. 5. Do you predict that photosynthetic rates will be higher or lower for dark-treated leaves? 4

SET-UP EXPERIMENT #1: 1. Use paper punch to make 30 leaf disks. Make the punches relatively close together (so you don t run out of leaf) and avoid large leaf veins. Put the leaf disks into the large Petri dish. 2. Take 3 syringes and add 10 leaf disks to each. Tap the syringe so all leaf disks are near the tip, and then replace plunger. Push plunger to the 5 ml mark don t squish leaf disks! BE SURE TO READ STEP #3 BELOW CAREFULLY!!! 3a. For 2 syringes (with leaf disks), suck up 10 ml of sodium bicarbonate solution. The sodium bicarbonate solution should be at the 15 ml mark on the syringe (leaf disks and air are between 0-5 ml). Do not mix up this syringe with the syringe containing distilled water below! 3b. For 1 syringe (with leaf disks), suck up 10 ml of distilled water. The distilled water should be at the 15 ml mark on the syringe (leaf disks and air are between 0-5 ml). Do not mix up this syringe with the syringes containing sodium bicarbonate solution above! 4. Hold syringes upright and expel air bubbles, as practiced earlier (see page 3). 5. Press thumb or index finger over syringe tips and pull plunger to infiltrate. Note the bubbles erupting from leaf disks! (How cool is that?!) Maintain your hold on the plunger with finger still on the syringe tip until bubbling stops, or at least greatly slows (~10-30 seconds). 6. Release plunger and tap syringe to dislodge leaf disks. Set syringe pointing up on table (resting on the plunger) do not mix up distilled water and sodium bicarbonate syringes! Wait a few seconds to see if leaf disks sink. If any leaf disks are still floating, repeat steps #4-6 above. (If leaf disks are still floating after three infiltration attempts, please consult instructor.) 7. When all leaf disks have sunk, remove plunger and add contents to plastic cup. Be sure to note which cup contains the distilled water! If necessary, add additional sodium bicarbonate solution or distilled water to bring liquid to desired level for evaluating leaf disk flotation. (Please see note on table about the proper solution level.) 8. Proceed quickly with next steps to minimize light exposure. Please note that if you aren t able to proceed quickly with the assay, you should put cups in dark place until you're ready. 9. Put distilled water cup and 1 sodium bicarbonate cup under light. Bend neck of lamp so that it is the proper distance from cups. (Please see note on table about proper height of light above the cups). Confirm that both cups are for sure getting comparable amounts of light. 10. Put 1 sodium bicarbonate cup in dark cupboard at lab table. 11. Start timer. Every minute count the number of disks that are floating and record data in Table 1 (page 6). Just before making your count, gently swirl cup to dislodge any disks that are stuck together. Be sure to minimize the time the dark treatment is exposed to light. 12. Continue your assay until all disks float or for 20 minutes, whichever comes first. 13. Remove dark treatment and dispose of liquid and leaf disks. Please use strainer at sinks to capture leaf disks so that we do not clog the drains! 5

14. Put sodium bicarbonate light treatment in dark cupboard and set timer for 10, 20, & 30 minutes. Record floating disk number for each time period in Table 2 (page 6). 15. Graph data from Table 1 on the template (see page 7). TABLE 1. Effects of CO 2 and light treatments on photosynthesis. Please record the number of leaf disks floating at each measurement time (1-20 minutes or until all disks floating). No. Minutes Sodium bicarbonate + light treatment Distilled water + light treatment Sodium bicarbonate + dark treatment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 TABLE 2: Results of sodium bicarbonate + light treatment container placed in dark cupboard following experiment. Please record the number of leaf disks floating. Minutes 10 20 30 Sodium bicarbonate + light treated container placed in dark cupboard 6

Questions about carbon dioxide levels and light: Use the above graph to answer the questions on the next page of the handout! 7

1. Through the process of photosynthesis, ivy produces oxygen bubbles that become trapped in the spongy layer of the leaf. Where did this oxygen originate? The time required for 50% of the leaf disks to float is called ET 50 ("effective time 50"). Because some leaf disks may behave weirdly and float earlier or later than others, it is often more meaningful to focus on this median time to float rather than total time required for all disks to float. Note that smaller ET 50 values indicate higher rates of photosynthesis (i.e., the more rapidly leaf disks float in this assay, the more photosynthesis is taking place in the leaf disks). 2. Inspect your graph and identify the ET 50 values for the three experiment treatments. 3. How did CO 2 addition affect photosynthesis? (Use ET 50 values to answer.) 4. How did light affect photosynthetic rates? (Use ET 50 values to answer.) 5. When a cup with floating leaf disks is placed in the dark, what happens? Interpret this finding. (Observe the sodium bicarbonate + light cup with floating disks that you placed in the dark; you will need to wait at least 30 minutes to answer this question.) 6. Does photosynthesis ever occur in leaf disks placed in distilled water? Interpret this finding. (Observe the distilled water cups placed under lights on the side bench; note how long the various cups have been under light, and how many disks are floating.) 8

Experiment #2: Work with your lab partners to design your own experiment! Now you will design an experiment to test other factor(s) that affect photosynthesis. The following items are available for your use. Ivy leaves of different ploidy levels (diploid Hedera helix and tetraploid H. hibernica) Ivy leaves of different ages Ivy leaves that have been exposed to light or that have been kept in the dark Solutions with different levels of sodium bicarbonate Solutions with different temperatures Food coloring for different colored solutions Fluorescent and LED bulbs Blocks to position leaf disks at variables distances from light source As a group, discuss the factor(s) you would like to test and how you will do this using available supplies and the lab set-up. (Remember that there is space for only three leaf disk containers under the lamps, though you could accommodate additional containers if they don't use a lamp treatment.) If you want something that isn t on the list, ask we may be able to find it for you. Fill out the table below with your observation, question, prediction, method, and control, following the format used above on pages 3 and 4. When you have fully thought through your experiment and completed the table, touch base with your instructor to confirm their approval for your approach. Motivation and Design of Student Experiment Observation: Question: Method: Control: 9

Proceed with your experiment and record your data in the table below. Be sure to identify the factor(s) being tested in the table and column headings! TABLE 3: Student-designed experiment evaluating effect of on photosynthesis rates in ivy leaf disks. No. Minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 After completing the above data table for your experiment(s), return to page 7 of the handout to graph your findings in the template provided. Now answer questions below about results of your experiment. As a group, you will present this information to your instructor; everybody should be active in this process! 10

1. What factor(s) related to photosynthesis did you choose to study? 2. What was the motivation to study this factor? How does the factor you tested apply to the real world (i.e., what might cause this factor to vary in nature and affect ivy)? 3. What was your prediction? 4. What was the method you used to test the prediction (e.g. variables and control)? 5. Using the graph of your data on page 7, summarize your results. How did your treatments affect photosynthetic rates? How did this compare to results of the control? 6. Did you get the predicted result in the experiment? If so, what does this confirm about your understanding of photosynthesis? If not, what may explain the unexpected results? 7. What potential sources of error were present in this experiment? How would you change the experimental design if you were to run this experiment again? 11