Laboratory 14: Photosynthesis

Objectives: After completing this laboratory exercise, you should be able to:

• Successfully utilize paper chromatography to identify various plant pigments

Develop a hypothesis regarding the factors that affect the rate of photosynthesis that you will test in order to evaluate your hypothesis

• Explain the effect of different light wavelengths on the rate of photosynthesis

The evolution of photosynthesis changed life on planet Earth in many profound and irreversible ways. Like cellular respiration, photosynthesis is an ancient biochemical process, with evidence in some bacterial groups approximately
3.4 billion years ago. Another group known as cyanobacteria became common about a billion years later. Like cellular respiration, huge quantities of ATP and electron carriers are involved. Like cellular respiration, photosynthesis is essentially a redox reaction, where one molecule loses electrons and another molecule gains them.

So how does photosynthesis differ from cellular respiration? In photosynthesis, carbon dioxide gets reduced to a simple sugar, and water (H2O) gets oxidized to become H+ and molecular oxygen (O2). In eukaryotic cells, photosynthesis also differs by where it occurs. Whereas cellular respiration takes place in both the cytoplasm and throughout the mitochondria, photosynthesis only occurs inside a chloroplast. And of course, photosynthesis is driven by light energy reacting with pigment molecules (chlorophylls mostly). Here’s a summary equation of photosynthesis:

The lab on cellular respiration should have provided insight into factors that could influence the rate of any enzymatic reaction. Using what you learned from that lab along with what you’ve discovered from your chapter readings and your lecture material, write down some of the factors that you think could influence how quickly the reaction summarized above would proceed. List at least three factors below:
1.

2.

3.

4.
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Part 1. Understanding light absorption

An absorption spectrum indicates the relative amount of light absorbed across a range of wavelengths. The graphs above represent the absorption spectra of individual pigments isolated from two different organisms. One of the pigments is chlorophyll a, commonly found in green plants. The other pigment is bacteriorhodopsin, commonly found in purple photosynthetic bacteria. The table above shows the approximate ranges of wavelengths of different colors in the visible light spectrum.

(a) Identify the pigment (chlorophyll a or bacteriorhodopsin) used to generate the absorption spectrum in each of
the graphs above. Explain and justify your answer.

(b) In an experiment, identical organisms containing the pigment from Graph II as the predominant light capturing pigment are separated into three groups. The organisms in each group are illuminated with light of a single wavelength (650 nm for the first group, 550 nm for the second group, and 430 nm for the third group). The three light sources are of equal intensity, and all organisms are illuminated for equal lengths of time. Predict the relative rate of photosynthesis in each of the three groups. Justify your predictions.

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Part 2. Quantifying light reactions and the rate of photosynthesis

OVERVIEW
Oxygen (O2) is one of the byproducts of photosynthesis. The gas forms as H20 molecules lose their electrons to the light-sensitive pigments. We can use this formation of O2 to investigate the factors that influence the rate of photosynthesis.

Preparation

Each group will need a collection of plastic cups filled with bicarbonate solution, several dozen spinach leaf discs that you will prepare using fresh spinach leaves and a cork boring tool, a light source and a hypothesis to test.

1. On the first page of this lab, you were asked to write down three factors that could influence the rate of
photosynthesis. In collaboration with your lab mates, select one of these factors that you would like to test that can be completed with the supplies on hand and in the time allotted for this exercise (about 30 minutes). Write down your hypothesis in the space below:

2. Now design an experiment that will test your hypothesis. Identify your independent and dependent variables, and
specify your control treatment. Outline your experiment in the space below before starting any procedures. The information and answers generated in Part 1 can be of tremendous help in guiding your design. If you are unsure, discuss your design with your instructor.

As part of your experiment, draw a graph (next page) of what your data should look like if your hypothesis were to be supported by your experimental results. You are making a prediction here, before actually conducting your experiment. Be sure to label all components of your prediction graph properly.

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Now you are ready to set up your experiment.

1. Fill each cup required for your experiment with bicarbonate solution. The volume used should be the same for all
cups (about 30-40 mL). The number of cups required will vary by group, depending on the hypothesis being tested. Each group should plan on at least three cups.

2. After cutting a generous supply of leaf discs all of the same diameter (unless this is part of your experimental
design) -take care to avoid major leaf veins – you will need to vacuum extract all air out of the mesophyll layer of the discs so that they remain submerged in bicarbonate solution until you are ready to energize the leaves with light. Once energized, oxygen will begin to form and thus cause the leaves to float. We will use leaf flotation over time as a proxy for the rate of photosynthesis. Your instructor will demonstrate how to vacuum extract the air from the leaf discs using the 60 mL syringe and bicarbonate solution. Keep vacuum extracted, submerged leaves away from any light source until you are ready to initiate your experiment.

3. Gently transfer 15 to 20 leaf discs into each cup, keeping the number of discs constant for all cups. If any discs
start to float, discard them. Only use submerged, undamaged leaf discs for the best results, making sure they form a single layer at the bottom of the cup. Once all cups have been filled with bicarbonate solution and the appropriate supply of leaf discs, you can turn on your overhead lamp(s) and begin collecting data.

4. Every two minutes, you need to count the number of floating leaves and record these data.

5. Terminate your experiment after 30 minutes or once all leaves in at least one of your treatments are floating.
Graph your results and be sure to label all axes.

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Spinach leaf data Number of discs floating

Time interval
Treatment
Treatment
Treatment
Treatment

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Discussion questions:

1. Compare your actual results from Part 2 with your initial prediction. Describe any similarities or
differences you see.

2. Did you find evidence in support of your hypothesis?

3. If you were to repeat this experiment, what improvements would you make to boost your confidence in
your data?

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Part 3. Paper Chromatography

Photosynthetic organisms use a mix of light-sensitive pigments. When light strikes these pigments, electrons can be stripped from water molecules. These electrons are then passed along from one protein complex to the next generating a flow of current that can be used to synthesize ATP in an electron transport chain. This exercise allows us to identify these pigments and explore some of their properties using paper chromatography.

1. Cut a strip of filter paper to 2 cm x 8 cm. Draw a horizontal line with a sharp pencil (not pen) about 1.5 cm
from the bottom of your filter strip.

2. Now place a fresh spinach leaf on the line you drew and roll a coin over it so that you get a line of green
pigment on the filter. Repeat this step at least 8 times so that the green pigment line is dark and dense, but not smeared beyond the original pencil line. Each time you roll the coin over the line, use a fresh part of the leaf.

3. Use your ruler to measure the appropriate length of filter paper needed to hang from the inside edge of the
beaker to just above the bottom of the beaker. Fold the top of the strip over the rim of the beaker and secure it with a small piece of tape, or use a wooden dowel to suspend the filter strip into the beaker.

4. Now pour 5-10 mL of acetone or 70% isopropanol or into the small beaker, taking care not to wet the line of
pigment. The pigment line should now be suspended just above the solvent, with the bottom part of the filter paper fully immersed in the solvent. Use a larger beaker inverted over the small beaker to form a “chamber”. The solvent will move via capillary action to form a wetting front (or solvent front). As the solvent moves past the pigment, molecules will be carried along. Because each type of pigment molecule has a different molecular mass, charge and other physical properties, they will travel at different rates and you will eventually see separate bands of color form.

The ratio of the distance traveled by a compound to that of the solvent front is known as the Rf value; unknown compounds may be identified by comparing their Rf values to the Rf value of a known standard. To calculate the Rf for each pigment after one hour, use your ruler to measure the distance traveled for each pigment and the distance of the total solvent front:

Rf = distance traveled of pigment ∕ distance of solvent front

Record your data below.

Pigment Band Color Distance Traveled (mm) Rf
Solvent Front N/A 1.0
1) Carotene Yellow / yellow orange
2) Xanthophyll Yellow
3) Chlorophyll a Bright green / blue green
4) Chlorophyll b Yellow green / olive green
5) Other

YOU MAY OR MAY NOT OBSERVE ALL THE COLORS LISTED

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