163 Photosynthesis
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BIOLOGY LABORATORY LIGHT REACTIONS OF PHOTOSYNTHESIS
(Reviewed Fall 2008)
The process of photosynthesis consists of reactions that do not depend directly on light and others that do. The former constitute the Calvin cycle and include those steps in which carbon dioxide is assimilated by green tissues and converted into various organic compounds such as carbohydrates and amino acids. The "light reactions", on the other hand, comprise those steps in which the energy from visible light is captured by the photosynthetic pigments of plants and converted into chemical energy. The primary products of the lightdependent reactions are ATP, NADPH, and O2 (Figure 1). Once formed, ATP and NADPH are then used for the Calvin cycle. The O2 formed during the light reactions eventually is released into the air, thus replenishing the supply of molecular oxygen present in the atmosphere. Photophosphorylation, photoreduction of NADP+, and evolution of O2 during photosynthesis rank as three of the most important biological processes known. Both the light reactions and the Calvin cycle occur in chloroplasts. The Calvin cycle, including the fixation of carbon dioxide, takes place in the stroma of chloroplasts. In contrast, the light reactions, including O2 evolution and ATP and NADPH formation, occur in association with the thylakoid membranes. This type of compartmentalization within chloroplasts seems consistent with the generalization that electron transport chains in biological systems are localized on or within membranes. Because of this, CO2 fixation may be demonstrated using isolated chloroplasts, while the light reactions (photosynthetic production of NADPH and O2) may be demonstrated using only fragments of the internal membranes.
NADP + acceptor acceptor eLIGHT eH+ pump ephotosystem I photosystem II eH2O H+ 1/2 O 2 + 2H +
Electrochemical gradient used for ATP production
eNADPH
HIGH ENERGY
eLIGHT
LOW ENERGY
FIGURE 1: Schematic representation of electron flow in the light dependent reactions.
Photosynthesis EXPERIMENTAL DESIGN
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When a chloroplast preparation is exposed to light in the presence of an electron acceptor, reduction of the electron acceptor occurs (Figure 1). You will use the dye 2,6-dichlorophenolindophenol (DCPIP--0.01g DCPIP, 0.3g KH2PO4, 0.3g MgCl2 in 200 ml dH2O, pH 7.5) as an artificial electron acceptor in the place of the physiological acceptor NADP+. In its oxidized form, DCPIP is blue and absorbs light at 600 nm; whereas, in its reduced form, it is colorless and does not absorb light at this wavelength. Therefore, a decrease in absorbance at 600 nm is a measure of DCPIP reduction to DCPIPH2. You are responsible for designing experiments to investigate aspects of DCPIP reduction by isolated chloroplasts. Table 1 has been provided to guide you in this endeavor--the first line has been filled out as a suggested "baseline" from which to design additional studies. Part 1 Provide evidence that the change in absorbance described above is the result of DCPIP reduction by isolated chloroplasts in the presence of light. To do this, you should be able to answer the following questions: A. Does exposing DCPIP to isolated chloroplasts in the presence of light result in a change in absorbance in the sample? If so: B. Is light required for the reduction of DCPIP? C. Are chloroplasts necessary for the reduction of DCPIP? D. Is it possible that something besides DCPIP reduction is responsible for the change in absorbance in the sample? Part 2 Design a study to thoroughly investigate at least one factor that may influence the reducing power of isolated chloroplasts. Be sure to state your experimental question clearly and develop a hypothesis before you begin. In addition to the basic supplies at your table, other equipment available to you in the lab includes: rulers/meter sticks light meters clocks/stopwatches light filters of various colors tap water/water baths/incubators of various temperatures pH meter with concentrated HCl and NaOH (for adjusting pH)
Your instructor may be able to assist you in locating other equipment you may wish to use. Here are a few additional tips to guide you in your design: Plan ahead, but don't be afraid to "tweak" your design initial if results are unexpected. Try to examine your chosen variable across a broad spectrum. Don't forget the importance of replication. A single trial for each treatment is not particularly convincing. Consider sharing chloroplast preparations with other groups. Be careful not to manipulate multiple variables at the same time.
Photosynthesis TABLE 1: Composition and Incubation Conditions of Experimental Tubes Tube Incubation Conditions 30 cm from lamp, 3 min DCPIP 2.0ml Chloroplast 0.1ml (100l) H2O 2.8ml
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1 2 3 4 5 6 7 8 9 10 11 12
PROCEDURE A. Isolation of Chloroplasts from Spinach Leaves
The isolation of chloroplasts from spinach leaves should be carried out at 0-4oC and in the dark. Maintain the preparation at this temperature by appropriate use of ice buckets, prechilled solutions, and prechilled glassware. 1. Remove the central ribs of approximately 10 grams of spinach leaves (a little more than a handful). Weigh 10 grams of the de-ribbed leaves. 2. Cut the leaves into smaller fragments and transfer to a prechilled mortar containing 30 ml of grinding medium (0.4M sucrose, 50 mM Tris Buffer, pH 7.5) and a "pinch" of sea sand. Grind with a pestle for approximately two minutes, then strain and squeeze (with hands) through two layers of cheesecloth, allowing the filtrate to flow through a funnel into a prechilled beaker. 3. Pour the filtrate into two prechilled centrifuge tubes, making sure that the tubes contain approximately equal amounts of the green fluid. 4. Centrifuge the homogenate at approximately 1,600 rpm in a tabletop centrifuge for 2 minutes. 5. Pour equal amounts of the supernatant (contains chloroplasts, thylakoid membranes, and other cell components) into two prechilled centrifuge tubes, centrifuge them at approximately 2,400 rpm in the tabletop centrifuge for 5 minutes. 6. Pour off and discard the resulting supernatant (contains broken chloroplasts, mitochondria, various membrane fragments, and soluble components). 7. Add 1 ml of grinding medium to each tube containing the pellet (contains whole chloroplasts). Resuspend the pellet using a clean transfer pipette. Combine the chloroplast preparations into one test tube. Keep this suspension on ice and in the dark..
Photosynthesis B. Reduction of DCPIP under differing experimental conditions
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NOTE that in this study you are measuring a DECREASE in absorbance, as opposed to an INCREASE in absorbance (as in previous studies). Carefully read and follow all instructions for calibrating and using the spectrophotometer! NOTE that each tube must be prepared and run individually. DO NOT add the chloroplast suspension until you are ready to begin a treatment! 1. Set the wavelength control on the spectrophotometer to 600 nm. 2. With nothing in the sample well, set the absorbance to infinity with the left hand control knob. 3. Prepare an experimental tube, add the chloroplast suspension, mix by inverting the tube while covering the opening with a piece of Parafilm and your thumb, and immediately place the tube in the sample well of the spectrophotometer. 4. Set the absorbance of this tube to 1.0 with the right hand control knob. 5. Remove the tube and position it at the desired distance from the front of a growth lamp. 6. Turn on the lamp and illuminate the sample for the desired amount of time. 7. Immediately re-mix the sample, place the tube in the sample well, and read and record the absorbance at 600 nm. Has a reduction of DCPIP taken place? How do you know? 8. Repeat the above procedures for the rest of your tubes. For each tube, obtain readings at 600 nm immediately before and after periods of illumination.
D. Presentation At the next laboratory, your group should be prepared to orally present the study you designed and conducted in Part 2 above. Your presentation should not exceed five minutes and length, and must include: Clear statement of your purpose and hypothesis. Summary of experimental treatments you applied. Visual presentation and summary of results. Conclusion(s).
Use of Powerpoint is strongly recommended. Computer video projection will be available for your use.
____________________ Based on a laboratory in Bregman, Allyn.1990. Laboratory Investigations in Cell and Molecular Biology, 3rd. Ed., Wiley, NY. pp. 165-179.