PART B The process whereby plants convert light energy into chemical energy is photosynthesis. The process occurs entirely within the chloroplasts. It can be summarized by the following equation: 6CO2 + 12H2O C6H12O6 + 6O2 + 6H2O This equation actually summarizes two sets of reactions — the LIGHT REACTIONS and the DARK REACTIONS. As their names imply, one reaction requires light, but the other does not. The light reactions convert solar energy into the energy needed (ATP and NADPH) by the dark reactions to convert carbon dioxide into carbohydrates. The HILL REACTION (also known as photolysis) is the phase of the light reactions in which electrons are split from water and transferred to an electron acceptor using the energy of the sun. These are the electrons that will be passed down an electron transport chain and be used to generate ATP via PHOTOPHOSPHORYLATION. At the heart of the light reactions are two PHOTOSYSTEMS, designated photosystem I and II. Each is composed of a collection of pigments, such as chlorophyll, carotenoids, and cytochromes, and other electron acceptors. Each photosystem has a different wavelength of light that it aborbs the best. Photosystem I absorbs maximally at 700 nm and photosystem II absorbs maximally at 680 nm. The electrons that are excited by this energy are transferred either to plastoquinone (PSII) or to ferredoxin (PSI). The two photosystems are linked by an electron transport chain (ETC) (refer to your text for details). Ultimately, the electrons are passed to NADP+, the oxidized form of nicotinamide adenine dinucleotide phosphate, to form NADPH. In 1937, Robert Hill showed that isolated chloroplasts can evolve oxygen in the absence of CO2. These results provided some of the first evidence that the source of electrons in the light reactions is water. Hill used an artificial electron acceptor that intercepted the electrons before they reached PSI, but after the major electron transport chain. The Hill reaction is formally defined as the photoreduction of an electron acceptor by the hydrogens of water, with the release of oxygen. The rate of the Hill reaction can be measured by the addition of an artificial electron acceptor that goes through a color change as it is reduced. DCIP, 2,6-dichlorophenolindophenol, is blue in its oxidized form and colorless in its reduced form. The color change can be measured using a spectrophotometer set at 600nm. The change in absorbance of the reaction mixture will be measured at 1 minute intervals to assay for the progress of the Hill reaction using chloroplasts that will be isolated from spinach leaves. Chloroplasts will be isolated by following a standard CELL FRACTIONATION protocol. Homogenized leaf tissue will be centrifuged to sediment cell debris first at low speed (200g), then chloroplasts second at a slightly higher speed (1300g). You will measure the normal rate of the Hill reaction and compare it to the rates in the presence of two inhibitors. One inhibitor is ammonia which is an UNCOUPLER, a compound that separates the process of photophosphorylation from electron transport. The ETC functions, but no ATP is produced. The uncoupler will cause an increased electron flow. The second inhibitor is an herbicide called DCMU (3-(3,4-dichlorophenyl)-1, 1-dimethylurea). DCMU blocks both electron transport and photophosphorylation by interrupting electron flow at the beginning of the ETC. Biology 3403 lab-Photosynthesis 3 PROCEDURE: Isolation of chloroplasts1. Place a clean glass beaker, two15 ml test tubes, and Tris NaCl buffer into the ice bucket to chill. 2. Remove the major veins from fresh spinach leaves until you have accumulated at least 4g of leaf material. 3. Cut the leaves into small pieces with scissors and place in a chilled mortar with 15 ml of ice-cold TrisNaCl buffer and a dash of purified sand. Grind the tissue with a chilled pestle for 2 minutes. The sand helps to prevent the rupture of chloroplasts during grinding. 4. Filter the suspension through 4 layers of cheesecloth into a chilled beaker. Transfer into a chilled 15 ml centrifuge tube. Wring out the remaining juice into the tube. 5. Centrifuge the filtrate at 200g for 1 min using the table top centrifuge. Be sure to balance your tube with another tube filled with the same total volume. 6. Decant the supernatant into a clean, chilled tube and spin at 1300g for 5 minutes. 7. Decant and discard the supernatant. Add 10 ml of ice-cold Tris-NaCl buffer to the pellet and gently resuspend the chloroplasts with a transfer pipet until they are thoroughly mixed. This is your CHLOROPLAST CONCENTRATE. 8. Remove 2 mls of the chloroplast concentrate and dilute with 5 mls of ice-cold Tris-NaCl buffer. Cover, label, and place the test tube on ice. This is your CHLOROPLAST SUSPENSION. You may now leave the Tris NaCl buffer out at room temp. Testing the chloroplast activity – These steps are designed to test the activity of the chloroplast (CP) suspension you have just isolated and to help guide decisions for dilutions that will work for the experimental tubes. The activity of every CP suspension is different and therefore this step is necessary. 1. Set the spectrophotometer for 600nm wavelength. 2. Add 150 ml of water to a 250 ml beaker. The temperature of the water bath will become warmer while the lamp is on and should be kept around 20°C by adding a FEW ice chips when needed. The reaction will be run in the water bath during illumination so that the temperature remains fairly constant. 3. Place the water bath 25cm from a lamp bulb (100 watt frosted incandescent). Keep the lamp off until the beginning of each trial. 4. Prepare the BLANK according to the Table below. Add the solutions in the sequence given across the top of the table from left to right. Mix the chloroplast suspension before adding it to the BLANK. Use parafilm to cover the top and invert the cuvette once. Use the blank to ZERO your spectrophotometer. 5. Prepare Positive Control ONLY. Add the solutions in the sequence given across the top of the table from left to right. Mix the chloroplast suspension before adding it to the reaction mix. Use parafilm (at front table) to cover the top and invert the cuvette once. 6. Quickly place the positive control tube into the water bath, turn on the lamp, and note the time. After 1 minute illumination, remove the tube from the water bath and QUICKLY invert and wipe the surface of the cuvette and measure the absorbance. All absorbance readings should be taken as quickly as possible because the DCIP will begin to revert to its oxidized (blue) state as soon as the chloroplasts are removed from the light path. Biology 3403 lab-Photosynthesis 4 7. Return the positive control tube to the water bath and continue to take absorbance readings at 1 min intervals for 10 minutes. If there are plenty of functional chloroplasts, the absorbance should reach zero (or close to it) in 7-10 minutes. 8. If your results reached zero in 7-10 minutes you can skip to the next section. If not, determine which kind of adjustment should be made to your chloroplast (CP) suspension. a) absorbance dropped too quickly (close to zero before 6 minutes) – dilute your CP suspension with Tris-NaCl buffer (start with 1:1 or 1:3 for example) and go back to step 4 (prepare a new blank with the new chloroplast mixture) b) absorbance dropped too slowly (didn’t reach zero in 10 minutes) – use the undiluted CP concentrate and go back to step 4 (prepare a new blank with the new chloroplast mixture) 9. Once you have determined the best concentration to use, LABEL this tube and keep it on ice for the remaining experiments.1. Use Excel to graph the reaction rate (as change in absorbance) over time and attach it. All treatments should be displayed on the same graph 2. Which tube resulted in the fastest reaction rate (steepest slope)? 3. What was the importance of the negative control? 4. Explain the curve that was produced for the ammonia treatment and compare it to the DCMU treatment. Are these results consistent with what is known about the action of these two chemicals with respect to electron transport? 5. Why is it important to take absorbance readings and return tubes to the light as quickly as possible?
