Photosynthesis lab

The data analysis file is here: photosynthesis.Rmd

Part 1: Effects of climate change on photosynthesis

Introduction:

We will use a whole punch to punch out small leaf disks. Normally leaf disks float. When the air spaces inside the leaf disk are filled/infiltrated with solution, the overall density of the leaf disk increases and the disk sinks. The infiltration solution includes a small amount of sodium bicarbonate. Bicarbonate ion serves as the carbon source for photosynthesis. As photosynthesis proceeds oxygen is released into the interior of the leaf which changes the buoyancy–causing the disks to rise. Since cellular respiration is taking place at the same time, consuming oxygen, the rate that the disks rise is an indirect measurement of the net rate of photosynthesis.

Objective:

We will measure the rate of photosynthesis occurring in spinach leaf disks. Next, YOU will change the environment around the leaf disks and measure the effects on photosynthesis.

Design an experiment to test 1 variable that affects the environment for the plant cells using the control described below. You can see the available materials listed below. You will have 4 beakers - 1 control and 3 to test your variable. Please run you ideas by your instructor before starting.

Materials:

• Plastic syringe (10 cc or larger)
• Spinach leaves
• Hole punch
• 4 - 250ml beakers
• Light source
• Hot plates
• Sodium bicarbonate (Baking soda) as carbon source
• Acetic acid pH3

Methods:

1. Prepare the 4 beakers:
   • Control: In the first 250ml beaker, add 150ml of 0.2% bicarbonate solution to the beaker and add 1 drop of dilute liquid soap to this solution to prevent leaf disks from sticking to glass.
   • Experimental: In the 3 experimental 250ml beakers, set up your climate change experimental design so that you end up with 150ml of bicarbonate solution and add 1 drop of dilute liquid soap to each.
2. Prepare spinach last! Have everything ready to go, then everyone works on spinach.
3. Cut 10 uniform leaf disks using a whole punch for each trial, cut 40 total. The leaf surface should be smooth and not too thick, avoiding major veins.
4. Infiltrate the leaf disks with the Sodium bicarbonate solution:
   • Remove the piston or plunger and place the leaf disks into the barrel of a syringe. Replace the plunger being careful not to crush the leaf disks. Push on the plunger until only a small volume of air and leaf disk remain in the barrel (< 10%).
   • Pull a small volume of Sodium bicarbonate solution into the syringe. Tap the syringe to suspend the leaf disks in the solution.
   • Hold the syringe vertically and slowly push in the plunger until just a bit of the infiltration solution is forced out–which reduces the amount of air space above the floating disks. This is an important step.
   • Holding a finger over the syringe-opening, drawback on the plunger to create a vacuum. Hold this vacuum for about 10 seconds. while holding the vacuum, swirl the leaf disks to suspend them in the solution.
   • Let off the vacuum. The bicarbonate solution will infiltrate the air spaces in the leaf causing the disks to sink.
   • You may have to repeat this procedure 2-3 times in order to get the disks to sink. Don’t overdo the vacuum procedure. Too much vacuum can damage the cells and cell spaces in the interior of the leaf and the procedure will likely not work. If you see the infiltration solution turning green you likely overdid the vacuum procedure.
   • Keep the sunk disc in the dark until ready to start the experiment.
5. Pour 10 disks into each of the 4 - 250ml beakers. You should have 10 disks in each beaker.
6. Start a stopwatch for each beaker when you expose the beaker to light. Record how long it takes for the first, second, etc. leaf to float to the surface.

Table 1: Time taken for disks to float

Analysis:

1. Why do the disks initially sink but eventually float?
2. Why might the median time to float be better than using an average time?
3. Determine the median time taken to float for your data. Since you have 10 disks, the median time is the time taken for 5 to float. Write the data in the table above.
4. The faster photosynthesis was happening, the lower the median time for the disks to float. Calculate the rate of photosynthesis by taking the reciprocal of the time (make sure you convert minutes & seconds into decimals). Write the data in the table above.
5. Which beaker of spinach disks had the fastest rate of photosynthesis?
6. Which beaker of spinach disks had the slowest rate of photosynthesis?
7. Based on the results of your experiment, how does understanding the rate of photosynthesis help us appreciate the role of plants in reducing CO₂ levels in the atmosphere?
8. What are some environmental factors that can affect the rate of photosynthesis?

Part 2: Identifying the activity of the Calvin Cycle

Phenol red is an organic dye that changes color depending it’s pH – that is, it is an indicator. It takes on a yellow color when in an acid, an orange-pink color at pH 7, and a dark pink color in a basic solution.

Since phenol red is an indicator, it is used to detect changes in pH. The pH of a solution can be changed slightly by the addition of CO₂. This produces carbonic acid, which is then converted to bicarbonate and hydrogen ions:

CO₂ + H₂O ⇌ H₂CO₃ ⇌ H⁺ + HCO₃^-

As more CO₂ is added, the reaction is driven to the right and it becomes more acidic. As you may have guessed, this change in acidity can be detected by the change in color of the Phenol Red. Similarly, if CO₂ is removed from the solution, the reaction will be driven to the left, and the acidity decreases.

As you have learned, the light-independent reactions of photosynthesis (the Calvin Cycle) essentially combine CO₂ molecules together with RuBP to form G3P, which in turn is combined to form glucose. As a plant photosynthesizes, CO₂ is used up from the solution, driving the reaction above towards the left, and increasing the pH.

Methods

1. Place a few drops of Phenol Red into tap water. This should make the tap water decidedly pink.
2. Using a straw, breathe air into the water. The air from your lungs has an elevated CO₂ content, and will acidify the water. Do this until the phenol red turns slightly yellow.
3. Divide the water into 2 equal parts and place in similar-sized beakers.
4. Place a sprig of elodea into one of the beakers.
5. Place both beakers in the sun, and leave them there for the duration of the lab.

Questions

1. Describe the initial color of both beakers.
2. Describe the color of the two beakers after about an hour in the sun.
3. What does the color indicate about the photosynthetic activity of the elodea? Be specific: you should be able to refer to the reactions of the Calvin Cycle.

Part 3: Determining the absorption spectrum of plant pigments

Each plant pigment absorbs a range of frequencies of light, with a particular frequency that it absorbs most efficiently. However, there are several pigments in leaf tissues. In this lab, we will investigate the light frequencies that are absorbed collectively by all the pigments present.

Methods

1. Cut one piece of plant material about 8cm long.
2. Boil plant in water (200 ml water in 600 ml beaker) for 2 minutes to soften the cell walls.
3. Transfer the boiled plant material to a beaker (100 ml) container containing 50 ml of 95% Ethanol.
4. Place the beaker containing the leaf and the ethanol in the boiling water bath for 5 minutes. At this point, you should have a beaker within a beaker.
5. Remove the inner beaker from the water bath and allow it to cool.
6. Filter the solution through a filter paper in a funnel into a small beaker.
7. Fill one cuvette with ethanol and another with the filtered pigments.
8. Set a spectrophotometer to measure % absorbance (ABS) and set it to 400nm.
9. Wipe the sides of the cuvette containing ethanol, insert it into the spectrophotometer, close the lid, and press CAL to calibrate the machine. Make sure you put the cuvette in properly. Ask me for instruction about how to do this.
10. Remove the cuvette with ethanol and put the cuvette with pigments in the spectrophotometer. Again, make sure it goes in the right way around! Record the absorbance.
11. Measure the absorbance at 25 nm intervals for the range 400 nm – 800 nm and enter your data in a table like the one below. You should re-calibrate the spectrophotometer every time you change the wavelength.
12. If absorbance is maxed out, dilute the sample and start again.

Data analysis

Use the photosynthesis.Rmd file. You will produce a plot in R of absorbance against wavelength. You may refer to figure 10.7 in your textbook except that you will only produce one absorption spectrum (for all combined pigments).

What you should turn in

Run the data analysis file photosynthesis.Rmd which will help you draw 2 graphs. You will also need to answer a few simple questions in the Rmd document and turn in the knitted (html) file.