AP BIO Lab 4: Photosynthesis

BACKGROUND
Photosynthesis fuels ecosystems and replenishes the Earth’s atmosphere with oxygen.  The process of photosynthesis involves the use of light energy to convert carbon dioxide and water into sugar, oxygen, and other organic compounds. This process is often summarized by the following reaction:

6 H2O + 6 CO2 + light energy -->C6H12O6 + 6 O2

OBJECTIVES:  
-Learn a technique to determine photosynthesis rates
-Compare photosynthesis rates under various conditions.
-Explain why the rate of photosynthesis varies under different environmental conditions.

Photosynthesis/The Light Reaction

What could you measure to determine the rate of photosynthesis?
• Production of O2 (How many moles of O2 are produced for one mole of sugar synthesized?) or
• Consumption of CO2 (How many moles of CO2 are consumed for every mole of sugar synthesized?)

Because the spongy mesophyll layer of leaves (shown in Figure 1) is normally infused with gases (O2 and CO2), leaves — or disks cut from leaves — normally float in water. What would you predict about the density of the leaf disk if the gases are drawn from the spongy mesophyll layer by using a vacuum and replaced with water? How will that affect whether or not the leaf floats? If the leaf disk is placed in a solution with an alternate source of carbon dioxide in the form of bicarbonate ions, then photosynthesis can occur in a sunken leaf disk. As photosynthesis proceeds, oxygen accumulates in the air spaces of the spongy mesophyll, and the leaf disk will once again become buoyant and rise in a column of water. Therefore, the rate of photosynthesis can be indirectly measured by the rate of rise of the leaf disks. However, there’s more going on in the leaf than that! You must also remember that cellular respiration is taking place at the same time as photosynthesis in plant leaves. (Remember that plant cells have mitochondria, too!) What else could be going on that might affect this process? Aerobic respiration will consume oxygen that has accumulated in spongy mesophyll. Consequently, the two processes counter each other with respect to the accumulation of oxygen in the air spaces of the spongy mesophyll. So now you have a more robust measurement tool —the buoyancy of the leaf disks is actually an indirect measurement of the net rate of photosynthesis occurring in the leaf tissue.

When immersed in water, oxygen bubbles are usually trapped in the air spaces of the spongy mesophyll in the plant leaf. By creating a vacuum in this experimental procedure, the air bubbles can be drawn out of the spongy mesophyll, and the space is refilled by the surrounding solution. This allows the leaf disks to sink in the experimental solution. If the solution has bicarbonate ions and enough light, the leaf disk will begin to produce sugars and oxygen through the process of photosynthesis. Oxygen collects in the leaf as photosynthesis progresses, causing the leaf disks to float again. The length of time it takes for leaf disks to float again is a measure of the net rate of photosynthesis. This process is shown in Figure 3.

In other experiments, a blue dye (2,6-dichlorophenol-indophenol, or DPIP) can be used to replace NADPH in the light reactions. When the dye is oxidized, it is blue. When reduced, however, it turns colorless. Since DPIP replaces NADPH in the light reactions, when the light strikes the chloroplasts, electrons boosted to higher energy levels through photosynthesis will reduce DPIP and turn it from blue to colorless. Chloroplasts will be extracted from spinach leaves and incubated with DPIP in the presence of light. As the DPIP is reduced and becomes colorless, the resultant increase in light transmittance is measure over a period of time using a colorimeter.

PROCEDURE: (This procedure is to test effect of CO2 vs no CO2 in water on rate of photosynthesis... MODIFY the procedure for your purpose question)
Step 1 Prepare 300 mL of 0.2% bicarbonate solution for each experiment. The bicarbonate will serve as a source of carbon dioxide for the leaf disks while they are in the solution.

Step 2 Pour the bicarbonate solution into a clear plastic cup to a depth of about 3 cm. Label this cup “With CO2.” Fill a second cup with only water to be used as a control group. Label this cup “Without CO2.” Throughout the rest of the procedure you will be preparing material for both cups, so do everything for both cups simultaneously.

Step 3 Using a pipette, add one drop of a dilute liquid soap solution to the solution in each cup. It is critical to avoid suds. If either solution generates suds, then dilute it with more bicarbonate or water solution. The soap acts as a surfactant or “wetting agent” —it wets the hydrophobic surface of the leaf, allowing the solution to be drawn into the leaf and enabling the leaf disks to sink in the fluid.

Step 4 Using a hole punch, cut 10 or more uniform leaf disks for each cup. Avoid major leaf veins. (The choice of plant material is perhaps the most critical aspect of this procedure. The leaf surface should be smooth and not too thick.)

Step 5 Draw the gases out of the spongy mesophyll tissue and infiltrate the leaves with the sodium bicarbonate solution by performing the following steps:   
a. Remove the piston or plunger from both syringes. Place the 10 leaf disks into each syringe barrel.
b. Replace the plunger, but be careful not to crush the leaf disks. Push in the plunger until only a small volume of air and leaf disk remain in the barrel (<10%).
c. Pull a small volume (5 cc) of sodium bicarbonate plus soap solution from your prepared cup into one syringe and a small volume of water plus soap into the other syringe. Tap each syringe to suspend the leaf disks in the solution. Make sure that, with the plunger inverted, the disks are suspended in the solution. Make sure no air remains. Move the plunger to get rid of air from the plunger before you attempt Step d.
d. You now want to create a vacuum in the plunger to draw the air out of the leaf tissue. This is the most difficult step to master. Once you learn to do this, you will be able to complete the entire exercise successfully. Create the vacuum by holding a finger over the narrow syringe opening while drawing back the plunger (see Figure 6a). Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the leaf disks to suspend them in the solution. Now release the vacuum by letting the plunger spring back. The solution will infiltrate the air spaces in the leaf disk, causing the leaf disks to sink in the syringe. If the plunger does not spring back, you did not have a good vacuum, and you may need a different syringe. You may have to repeat this procedure two to three times in order to get the disks to sink. (If you have any difficulty getting your disks to sink after three tries, it is usually because there is not enough soap in the solution. Try adding a few more drops of soap to the cup and replacing the liquid in the syringe.) Placing the disks under vacuum more than three times can damage the disks.

Step 6 Pour the disks and the solution from the syringe into the appropriate clear plastic cup. Disks infiltrated with the bicarbonate solution go in the “With CO2” cup, and disks infiltrated with the water go in the “Without CO2” cup.

Step 7 Place both cups under the light source and start the timer. At the end of each minute, record the number of floating disks. Then swirl the disks to dislodge any that stuck against the side of the cups. Continue until all of the disks are floating in the cup with the bicarbonate solution.

Step 8  Record data in table and calculate rate of photosynthesis by dividing number of disks floating by time it took them to float.