Topic 8. Exercises on Photosynthesis. I. Where Photosynthesis Occurs in Plant Cells

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1 Topic 8. Exercises on Photosynthesis The pathways of photosynthesis and respiration are quite different. However, at the global level, one is perfectly complementary to the other as the end products of respiration are the reagents for photosynthesis. The oxygenated air that sustains all animal life is due to photosynthesis. This fact was only discovered in the late eighteenth century when Joseph Priestly found that plants could fix air exhausted by fire and/or respiration to allow a mouse to survive in a closed jar. It is worth considering the overall reactions of photosynthesis and respiration. Respiration C 6 H 12 O O 2 > 6CO H 2 O Photosynthesis 6CO H 2 O > C 6 H 12 O O 2 Web Lesson@ I. Where Photosynthesis Occurs in Plant Cells While some parts of respiration occur outside the mitochondrion, all parts of photosynthesis occur in the chloroplast. Inside the chloroplast is an internal system of membranes called thylakoids, some of which are clustered into stacks called grana. The space not occupied by these membranes is called the stroma. The Light Dependent Reactions The light reactions require the spacial structure generated by the intact thylakoids. The pigment systems of light reaction I and II are both bound to these membranes and are each coupled with an electron transport chain. As in the mitochondrion, these electron transport chains generate a hydrogen ion concentration across a boundary which is harnessed to generate ATP. The ultimate electron receptor in the process is not oxygen, as in the mitochondrion, but NADP+ which generates NADPH. The Light Independent Reactions The Calvin Cycle, which fixes carbon, occurs in the stoma. It requires the reducing potential (NADPH) and the energy (ATP) provided by the light reaction, but is not, itself, dependent upon the structure of the internal membranes of the chloroplast (other than to keep the whole mix together). To produce sugars and to evolve oxygen, the light reaction and the Calvin Cycle must work in tandem. While the light reaction can produce ATP without the Calvin Cycle, it cannot split water without the regeneration of NADP+ from NADPH accomplished by the Calvin Cycle. The Calvin Cycle, in turn, cannot fix CO 2 without a constant supply of ATP and NADPH from the light reaction. Consider the figure on the next page

2 1. Where do the light reactions occur? 2. Where does the Calvin Cycle occur? II. The Necessity of Chlorophyll for Photosynthesis. To eat light as plants do, it must first be absorbed. As plants are universally green due to chlorophyll, it seems obvious that a green pigment is necessary for photosynthesis. However, correspondence doesn t constitute proof of causality. The following is a simple exercise to provide evidence of that relationship. In this exercise we will consider the hypothesis

3 Chlorophyll is necessary for photosynthesis. At the side bench is a variety of Coleus with variegated leaves. There are two obvious pigments to be found in various regions of its leaves. Observe leaves on the plant, and note that some leaves have areas with... - just chlorophyll colored green; - just anthocyanin colored red; - regions of overlap, and - regions with neither which are white. Because chlorophyll is not found everywhere in the leaf, we can use this plant to evaluate whether chlorophyll is necessary for photosynthesis. To do so, however, we require a method for determining photosynthetic activity. One approach is to consider where in the leaf starch forms. Starch is derived from sugars produced by photosynthesis and we can detect starch using iodine in I 2 KI. Procedure: Work in pairs. 1. Take a leaf and draw it. Clearly indicate where chlorophyll and anthocyanin are located and where they overlap. 2. Boil the leaf in water to remove the water soluble anthocyanin pigment. Make a second drawing clearly showing where the chlorophyll is located. 3. Boil the leaf in alcohol to remove the chlorophyll pigmentation. 4. Carefully place the bleached and brittle leaf on a watch glass and flood it with I 2 KI. 5. Again draw the leaf clearly indicating where the purple stained starch is located. Drawing 1 Drawing 2 Drawing 3 Untreated leaf Anthocyanin removed Stained with I 2 K Discussion:

4 Are your results consistent with the hypothesis? Consider a second hypothesis: Anthocyanin is necessary for photosynthesis. Do your results support that statement? Can you reject the second hypothesis? Have you proved the first hypothesis? III. The Necessity of Carbon Dioxide and Light for Photosynthesis. This activity introduces a simple way of determining photosynthetic activity, to evaluate the two hypotheses: Carbon dioxide is necessary for photosynthesis. and Light is necessary for photosynthesis. Experimental Design All plant tissues contain intercellular spaces. Those in the leaf are filled with air, which allows for rapid diffusion of gases. They also make leaf tissue buoyant. If the air is removed, leaf tissue will sink. If the tissue is living and is then exposed to light, photosynthesis will generate O 2 which will refill the spaces with gas and cause the tissue to again float. In this exercise photosynthetic activity will be determined for three different experimental conditions as outlined below. 1. Light - discs in bicarbonate solution.

5 2. Light - discs in boiled distilled water. 3. Dark - discs in bicarbonate solution. For each condition a beaker with 10 non-floating leaf disks will be tested. The beakers will all sit on a piece of white paper. Procedure (To be in teams of six students). Each team to work in three groups. One group to start part a, while another starts part c, while the third starts part e. a. Punch out forty leaf disks from green Coleus leaves and place them into a syringe. Try to avoid the midrib and the major veins. b. Add 7 ml (7cc) of dilute detergent solution to the syringe. Insert the plunger, invert the syringe, and push out all the air. While covering the opening at the front with your thumb, pull the plunger back to create a vacuum. Maintain the vacuum for 10 seconds while agitating the syringe and then slowly release the plunger. Repeat this procedure for two to three minutes until most of the disks have sunk. Remove the plug and immerse the syringe in a large finger bowl of tap water. Slowly pull the plunger out, sucking the water into the syringe. After removing the plunger carefully pour the disks into the water. c. Two other people should heat 500 ml of bicarbonate solution to 35º C. d. Fill two 250 ml beakers with the NaHCO 3 solution heated in Part c. Fill another with 200 ml of boiled and stoppered distilled water and label it. Then, using forceps, add 10 disks to each of the three 250 ml beakers. Only use disks that are not floating and introduce them into the water edge on to prevent bubbles from being captured on their surfaces. e. During the preceding steps two members of the group should position two lamps 50 cm above the bench surface. f. Place one beaker with bicarbonate and the one with distilled water below the lights, Place the other beaker of bicarbonate on the bench and cover with foil. Turn on both lights. g Run the experiment for 40 minutes or until you observe eight or more discs floating in the illuminated beaker of bicarbonate. h. Record the number of floating discs for each condition in the table. Light + bicarbonate Dark + bicarbonate Light + Distilled water

6 7. In the balanced equation for photosynthesis, for every mole of CO 2 consumed, one mole of oxygen is produced. Why do we observe an increase in the volume of total gas for photosynthesis in water? 8. In the beaker of distilled water, was any form of photosynthesis occurring? If so what were its products? 9. What is the source of the oxygen generated? 10. What prevents the discs in the distilled water from producing oxygen? IV. The Light Harvesting Pigments of Photosynthesis In the following activities you will observe specifically which colors of light are absorbed by a pigment extract from banana leaf, and, hence, which colors are used by photosynthesis. IVa. The Spectrum Viewer Work with a partner See the illustration of the spectrum viewer on the next page. These are located on the side bench. Look through the front opening. If the light behind the viewer is on and is properly aligned with the opening at the back, you should view a spectrum of colors projected to the right. Note that this spectrum is projected onto a scale. The scale is to be read in nanometers. Note that each color is projected at the point on the scale corresponding to that color s wavelength in nanometers.

7 Spectrum Viewer Illustrations Spectrum Viewer Look to the right to view the scale The spectrum from the light source will be projected against a scale in nanometers. Only push the filters and pigment extract half way down the opening at the back

8 Procedure: 1. Take color filters and push them half way down the opening at the back as you view the projected spectrum (see illustration). Note, if properly positioned you will still view the total spectrum of the light source on the bottom of the scale while also viewing the light transmitted through the filter at the top. Are the filters totally effective? - With the red filter do you see colors other than red? - With the green filter do you see colors other than green? - With the blue filter do you see colors other than blue? 2. Take a test tube of pigment extract, and, while viewing the projected spectrum, push the tube half way down the opening in the back. Carefully note that there are two regions where the pigment absorbs the light most completely. If you cannot discern these regions, the pigment may be worn out and will need to be replaced. If you have trouble here ask your TA for help! - The two regions of maximum absorption corresponds to what two colors? - The two regions of maximum absorption corresponds to what two ranges of wavelengths? IVb. The Spectrophotometer: Work in pairs. The spectrophotometer is on the side bench. Instructions for its use are provided by the instrument, and your TA is available to assist. With a spectrophotometer, we can quantify what you have just observed with the spectrum viewer. Each pair of students will measure the transmittance of light through a solution of pigment extract at each of the wavelengths given in the table. Transmittance is simply a measure of the portion of light not absorbed as passes through a sample. From your observations with the spectrum viewer it should be clear that this value will vary with color. The data table below will dictate your choice of wavelengths. Measure transmittance at each of these wavelengths. Record your readings below, and graph the data on the following page. In class, check with other students to verify that you didn t make mistakes. Turn in this table and graph in discussion.

9 Wave Length (nanometers) 400 Transmittance Do the results in Part VIb. Correspond to you observations in VIa? 2. Are they consistent with the information given in your book about the action spectrum of photosynthesis?

10 Name Partner % 70% 50% 30% 10%

11 IVc. Fluorescence of Chlorophyll If the light energy absorbed by chlorophyll is not transferred to a reaction center, then the energy is lost either as heat, or as fluoresced light, or some combination of the two. View the demonstration of fluorescence at the front bench. Do not confuse this fluoresced light with the transmitted light viewed by the spectrum viewer. What emits the green light viewed through the spectrum viewer? What emits the red, fluoresced, light? IVd. Isolating Pigments Using Paper Chromatography Work in groups of two The glassware required for this activity is at the front of your bench. The chromatography paper and solvent, and the pigment extract for use in this activity are at the front bench. Procedure: 1. Take a piece of chromatography paper, roll it into a cylinder, and staple it in three places (see illustration on the next page). 2. Remove the 500 ml lipless beaker from the larger of the two petri dishes (see illustration). Take the smaller petri dish and add enough pigment extract to cover the bottom. 3. Stand the paper cylinder in the smaller dish until the pigment has moved about 1.5 cm up the cylinder. 4. Set the paper aside for 5 minutes to dry. 5. Load the paper two additional times (repeat c and d twice). 6. After loading the paper with pigment the third time, allow to dry at least 10 minutes. Then return the pigment extract to the stock bottle; wipe the small petri dish with a tissue, half fill that dish with chomotography solvent and place it in the center of the larger petri dish; sit the paper cylinder in the pigment extract, and cover with the 500 ml lipless beaker. 7. If you properly dried your paper, you should quickly see the chromatography solvent move through the previously loaded pigment band with the immediate result that bands of pigment separate from each other.

12 Chromatography Glassware Chromatography Paper with smaller and larger petri dishes Explanation: These pigments are separating by their physical affinity for water (determined by the extent of their molecular polarity). Water is bound to the fibers in the paper, and as the chromatography solvent moves up the cylinder of paper, it acts as a drag on the pigment molecules. The extent of this drag is directly related to each molecule s attraction to water. At the top of the cylinder you should see a deep yellow band made up of the carotinoid pigment, carotene. This layer moves freely with the moving solvent front. It is non polar with no affinity for water. The next pigment down, represented by one or two yellow bands, is the carotinoid pigment, xanthophyll. Next going down the cylinder, is a green band made up of Chlorophyll a, and the lowest band is made up of the pigment with the greatest affinity for water, Chlorophyll b. Which of these pigments are integrated into the reaction centers of both photosystems I and II? Which act as accessory pigments in the antenna complexes of photosystems I and II?

13 Which serve to protect the photosystems from photo oxidative damage? Which is the plant source for vitamin A in our diet? V. C-4 Photosynthesis As discussed in your text, oxygen disrupts the Calvin cycle at the point of CO 2 fixation: RuBP carboxylase (Rubisco) catalyzes CO 2 fixation through a reaction between CO 2 and ribulose 1,5,-bisphosphate. However, this same enzyme will also catalyze a reaction between oxygen and ribulose 1,5,-bisphosphate. The reaction with oxygen is not productive and the biochemical pathway required to salvage the resulting product consumes oxygen and releases CO 2. This process is termed photorespiration. The name is deceiving as no ATP is produced. Photorespiration is particularly maladaptive in hot dry conditions where a plant s stomata are more likely to be closed. C-4 plants have evolved in response to this problem, and C-4 photosynthesis has evolved independently in many different lineages. Details of C-4 physiology are outlined in your text. Simply stated C-4 plants change the ratio of carbon dioxide to oxygen in the cells where the Calvin Cycle occurs. By increasing the level of CO 2 relative to O 2, the rate of photorespiration can be reduced. This is accomplished by first capturing CO 2 in a part of the leaf where the Calvin Cycle does not occur using the enzyme PEP carboxylase. This results in the formation of a four-carbon compound (hence the name C-4 Photosynthesis). This four-carbon compound is moved into another part of the leaf where it is broken down into a 3-carbon compound and CO 2. This CO 2 releasing reaction also produces NADPH from NADP+. The CO 2 and NADPH then enters into the Calvin Cycle. Procedure: On the side bench are corn plants. Corn is a C-4 plant. These plants were subject to 48 hours of continuous light. Under these the conditions, a leaf cell undergoing the Calvin cycle cannot export photosynthate as rapidly as it is produced resulting in the accumulation of starch in the cell. Hence, the presence of starch corresponds with where the Calvin Cycle occurred in the leaf. 1. Cut several leaf squares from the plant. Place these in a syringe with detergent water and subject the tissue to a vacuum to remove the air (your TA will demonstrate). These are ready for the next step when they loose buoyancy. 2. Pour your sunken squares into a communal bowl by the plants. Take a leaf square, place it on a microscope slide in a drop of water. Then, using a stainless

14 steel razor blades from the front of your bench, cut thin cross sections from the leaf square. This will be difficult. One approach for cutting thin sections is to make numerous choppy cuts and save only those cross sections narrow enough to be seen in profile. Another is to use two blades where one holds the leaf tissue down while the other makes the slice. One other technique is to place the leaf tissue between two sections of carrot and to make thin sections through the carrot tissue. Give yourself time as it may take several minutes before you generate a useful section. Ask you TA for help if you don t succeed after ten minutes or so of effort. 3. Make a wet mount and observe the leaf tissues in cross section. Pay close attention to the enlarged bundle sheath cells. Cross section of Zea leaf showing enlarged bundle-sheath cells 3. After making these observations treat your slide with I 2 KI by adding the stain to one side of the cover slip while soaking up water on the opposite side using tissue. 4. Again observe your leaf section. Make a quick sketch below showing where the starch is located.

15 5. In which part of the leaf is the starch located? This is also where the Calvin cycle occurs. 6. Observe the electron micrograph of corn leaf on the side bench and correlate your observations with it. Compare it with the ER of a C-3 plant. Why does the C-3 plant have more microbodies? Discussion Topics for Your Consideration 1. Consider the argument that the first instance of organisms polluting their environment were the Cyanobacteria releasing noxious diatomic oxygen. 2. How has the presence of oxygen gas made life on land possible?

16 3. As oxygen is cycled between respiration and photosynthesis, in what molecular forms is it found? 4. What came first, aerobic respiration or photosynthesis? 4. What came first, glycolysis or photosynthesis? On what basis can you evaluate the question? Web