biology 121 lab

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Objectives • Examine carbon fixation • Examine electron transport in

photosynthesis. • Examine the effects of inhibitors on

photosynthesis. Terms

Photosynthesis Biochemical reaction of photosynthesis Photochemical reaction of photosynthesis Photosynthetic electron transport chain Chlorophyll accessory pigments

Introduction Photosynthesis uses light energy to split H2O and harvest high-energy electrons. These energetic electrons (and accompanying H+) are passed to CO2. In doing so, CO2 is reduced to form energy-storing sugars. Cellular respiration removes electrons from (i.e. oxidizes) sugar, captures the energy in adenosine triphosphate (ATP), and ultimately passes the electrons to oxygen to form H2O. Organisms use the energy stored in ATP to conduct cellular business such as transport, synthesis of biomolecules, reproduction, and sometimes cellular movement.

LAB TOPIC 6: Photosynthesis

Remember that in photosynthetic eukaryotes (plants and algae), respiration occurs as it does in animal and fungal cells (Figure 6.1).

When in an aqueous solution, carbon dioxide reacts with water to form carbonic acid.

This results in lowering the solution's pH. As CO2 is consumed by aquatic plants through photosynthesis, the level of carbonic acid in a solution will decrease, leading to an increase in pH. Thus, monitoring pH provides an indirect measure of the amount of CO2 consumed in photosynthesis. This experiment uses bromothymol blue (BTB), a pH indicator that turns yellow at pH < 6.0, green at pH 6.0 - 7.6, and blue at pH > 7.6. We will be looking at changes in pH of water in the light and dark and presence and absence of the aquatic plant.

Exercise 6.1 Carbon dioxide fixation by an aquatic plant

Figure 6.1 Overviews of aerobic respiration and photosynthesis.

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Procedure 6.1 Carbon dioxide consumption

1. Place 75 ml of BTB solution into a 100 ml beaker. Blow exhaled air through a straw into the BTB solution until it changes from blue to yellow-brown.

2. Obtain two 2-cm sprigs of plant, and place one in a tube labeled "light" and the other "dark". The other two tubes will have no plant in them. Use these for comparison.

3. Fill the four test tubes 3/4 full with C02-rich BTB solution.

4. Place the two "light" tubes directly in front of the grow light, and the other tubes in the dark. .

5. Allow the tubes to "incubate" for 1 hour. Proceed to the next exercise while you wait. In Table 6.1, record any color changes that have occurred by marking an X in the appropriate space.

Hypothesis Construct null and alternative hypotheses for the effect of light and dark and presence and absence of a plant on pH. Remember, your hypotheses must be testable. Prediction Predict the results of the experiment based on your hypotheses. Your prediction would be what you expect to observe as a result of this experiment (if/then).

Yellow

pH < 6.0

Green

pH 6.0 – 7.6

Blue

pH > 7.6

plant, light

plant, dark

NO plant, light

NO plant, dark

Table 6.1 Carbon dioxide consumption by an aquatic plant

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Questions Do you accept or reject your null hypotheses?

Why did the BTB turn yellow as you blew through the straw?

What is responsible for the color change of BTB in the tube with the plant placed in front of the light?

Why might the tube with the plant placed in the dark exhibit an increase in pH?

Why might the tube with the plant placed in the dark exhibit a decrease in pH?

Exercise 6.2 Effects of herbicides on electron transport in

An overview of the light-dependent reactions of photosynthesis

In the late 1930’s, Robert Hill and colleagues observed that under proper conditions, isolated thylakoids retained their capacity to evolve oxygen. This phenomenon is now known as the Hill Reaction. The Hill Reaction is part of what are known as the photochemical reactions of photosynthesis. This activity is associated with Photosystem II (Figure 6.2), in which the electrons that originate with splitting of water are used to reduce electron acceptors. There is a simultaneous release of oxygen. In the intact living organism, these electrons ultimately reduce NADP+ to form NADPH. During photosynthetic electron transport, hydrogen ions are moved across the thylakoid membrane as plastoquinone shuttles electrons between PS II and the cytochrome B6/f complex. The resulting

hydrogen ion gradient is used to produce ATP. The ATP and NADPH then are used in the biochemical reactions to produce sugars, thus trapping light energy in the chemical bonds of carbohydrates.

Hill used artificial electron acceptors, including 2,6-dichloroindolphenol (DCPIP), to trap electrons passed through the electron transport chain from photosystem II when isolated chloroplasts are exposed to light. As the blue, oxidized form of DCPlP becomes reduced it becomes colorless. Thus the progress of the reaction can be monitored by the change in absorbance at 600 nm of the DCPIP solution.

DCPIP + 2H+ + 2e- → DCPIP-H2 (blue) (colorless)

(Remember that chlorophyll is green and so only the blue color will disappear entirely.)

The rate of the Hill Reaction is then dependent on light intensity and can be measured either as oxygen produced or reduction of electron acceptors.

First we will look at electron transport in thylakoids, then we will look at the effects of herbicides.

Figure 6.2 Photosynthetic electron transport

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Procedure 6.2.1 Electron Transport

1. Prepare test tubes according to Table 6.2. Metabolically active thylakoids will be provided to you. Add the DCPIP (blue dye) last.

2. Mix the contents of each tube by inverting each tube several times. Place tubes 2 and 3 in front of the light source for 2 minutes. Do not position tubes behind one another or in front of tubes from other groups. Place tube 4 in the dark.

3. Observe color changes in the tubes and

record your observations.

What were the initial and final colors of each tube? Of what importance is each tube (1-4) in this exercise? Which tubes were CONTROLS?

Table 6.2 Solutions for comparison of photosynthetic reaction rates

Tube Thylakoids 0.1 M phosphate buffer pH 6.5

Water 0.2 M DCPIP

1 0.5 ml 3 ml 1.5 ml 0

2-LIGHT 0.5 ml 3 ml 0.5 ml 1 ml

3 0 3 ml 1.0 ml 1 ml

4-DARK 0.5 ml 3 ml 0.5 ml 1 ml

Procedure 6.2. Effects of Herbicides on Electron Transport

Next we will monitor the effect of a photosynthetic herbicide on photosynthesis. We will quantify the electron transport (DCPIP reduction) using a spectrophotometer. Monitor absorbance at 600 nm. USE WATER AS A BLANK.

Hypothesis Construct null and alternative hypotheses for the effects of herbicides on electron transport in active thylakoids. Remember, your hypotheses must be testable.

Prediction Predict the result of the experiment based on your hypotheses. Your prediction would be what you expect to observe as a result of this experiment (if/then).

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1. Using water as a blank, calibrate your spectrophotometer.

2. Set up tube #1 as shown in Table 6.3.

3. Fill your cuvette with Solution 1 and place it in the spectrophotometer. Immediately, and at 1-minute intervals for 5 minutes, monitor absorbance readings at 600 nm.

4. Record your data in Table 6.4. Repeat steps 2 and 3 for tubes #2 and 3.

5. Plot your data using Excel, KGraph or the graph paper provided

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Table 6.3 Solutions for herbicide treatments

Tube Thylakoids 0.1 M phosphate buffer pH 6.5

Water or herbicide 0.2 M DCPIP

1 0.5 ml 3 ml 0.5 ml water 1 ml

2 0.5 ml 3 ml 0.5 ml herbicide A 1 ml

3 0.5 ml 3 ml 0.5 ml herbicide B 1 ml

Table 6.4 Absorbance data for herbicide treatments across time (T) in minutes

Tube T=0 T=1 T=2 T=3 T=4 T=5

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2

3

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Conclusions What happened when you illuminated the herbicide-treated thylakoids? Based on your observations, what can you say about the mode of action of each herbicide? Do you accept or reject your null hypotheses? What other set of oxidation-reduction reactions with electron transport might contribute to some of the color change in the BTB tube with the plant in the DARK? Under what circumstances might you measure carbon fixation in the dark?

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