Lab 3 Exploring Photosynthesis and Plant Pigments ADA PDF

Preview

Summary

Photosynthesis Lab emphasizes the idea of pigmentation and how substances change color and show how some things cause a reaction....

Description

BIOLOGY

Exploring Photosynthesis and Plant Pigments Investigation Manual

EXPLORING PHOTOSYNTHESIS AND PLANT PIGMENTS Table of Contents

Overview

2

Overview

2

Outcomes

2

Time Requirements

3

Background

7

Materials

8

Safety

Photosynthesis provides the energy for the fixation of carbon dioxide by photosynthetic organisms. CO2 is combined into sugars, which other organisms can consume. In this lab investigation, photosynthetic pigments will be isolated and separated from leaves of a green plant using paper chromatography. An additional experiment is conducted that looks at the effect that various conditions have on the production of oxygen as an indicator of photosynthesis.

8 9

Activity 1 Activity 2

Outcomes

12 Disposal and Cleanup 12 Extension Activities 13 Data Tables

• Determine the number of pigments in a sample using paper chromatography. • Formulate hypotheses predicting the effect of varying light conditions and dissolved carbon dioxide concentration on the rate of oxygen production as a proxy for the rate of photosynthesis. • Collect and graph data. Use the data to explain the need for light and carbon dioxide on the light-dependent reaction of photosynthesis.

Time Requirements Preparation ..................................................................... 20 minutes Activity 1: Plant Pigments Overview............................... 20 minutes Activity 2: Leaf Disk Assay Overview ........................... 120 minutes

Key Personal protective equipment (PPE) goggles gloves apron

follow link to video

photograph stopwatch results and required submit

warning corrosion flammable toxic environment health hazard

Made ADA compliant by NetCentric Technologies using the CommonLook® software

2

Carolina Distance Learning

Background All living things need a source of outside energy. Animals get their energy from the food they eat. Plants obtain this energy from sunlight and convert the energy into a usable form by a process called photosynthesis.

leaf surface called stomata (plural for “stoma”). The stomata allow gases such as carbon dioxide and oxygen to enter and exit the leaf. They also regulate the water content of the plant by evaporation by opening and closing.

Chloroplasts and Photosynthesis Plants, along with algae, cyanobacteria, and a few other organisms, perform photosynthesis using light energy to power a chemical reaction that yields energy, which can be stored as food.

Carbon dioxide, which is needed for the second part of the photosynthesis cycle, is found in the atmosphere and enters the leaf through the stomata, then diffuses into the mesophyll.

In plants, most photosynthesis occurs in the leaves, although some other parts of plants are also able to perform photosynthesis. Within the leaves, the mesophyll cells contain large numbers of chloroplasts. A cross section of a typical plant leaf (Figure 1.) reveals top, tight layers of palisade mesophyll cells. Below this is a lot of loosely arranged spongy mesophyll cells that allow for intercellular spaces. These spaces are continuous, with small openings in the lower

Light-Dependent Reactions of Photosynthesis In order to convert sunlight to chemical energy, plants contain a series of pigments to absorb sunlight. The most common of these are chlorophyll a and b and a group of pigments called carotenoids. These pigments are located clustered together and anchored by proteins in the antenna complex. In plants, this complex is located in the thylakoid membrane inside the plant’s chloroplasts. Each pigment has a distinct

Figure 1.

Figure 2.

continued on next page www.carolina.com/distancelearning

3

EXPLORING PHOTOSYNTHESIS AND PLANT PIGMENTS Background continued color dictated by the wavelengths of energy that it reflects. By contrast, the wavelengths that it absorbs are used to boost electrons to a higher energy state. As you can see in the picture, light that is not absorbed is reflected back. Thus a plant which looks green to us does not absorb the green light but reflects it back. In Figure 2 you can see that blue light is absorbed by the leaf. The energy absorbed is transferred to other pigment molecules via resonance energy transfer. In Figure 3 you can see the complex pathway the energy, in the form of an electron, will travel. After the electron enters the photosystem it will first enter photosystem II. This system has its own pigment, P680. The number indicates the wavelength the pigment absorbs, in this case

680 nm. P680 transfers its high-energy electron to the primary electron acceptor, pheophytin. The electron lost by P680 is replaced by the oxidation of water. The oxidation of water produces oxygen gas O2 and hydrogen ions, which will be used later in the photosynthesis process, to help drive the proton pump that produces ATP. 2 H2O → O2 + 4H ++ 4e− The high-energy electron lost by P680 to pheophytin is subsequently transferred to a molecule called QB, along with another electron and two hydrogen ions. QB is part of the electron transport chain. By moving through the electron transport chain, excited electrons and the cytochrome complex facilitate the movement of

Figure 3.

continued on next page 4

Carolina Distance Learning

hydrogen ions from the stroma to the thylakoid lumen, further increasing the electrochemical gradient across the membrane.

hydrogen ions in the stroma to NADPH, the final electron acceptor, further reducing the concentration of hydrogen ions in the stroma.

From the cytochrome complex, the high-energy electrons are passed to plastocyanin and then to another pigment molecule in photosystem I. The pigment associated with photosystem I (P700) reacts to a slightly different wavelength of light from the pigments in photosystem II (700 nm for photosystem I vs 680 nm for photosystem II). Each photosystem can work independently from the other, but receiving a high-energy electron from photosystem II, photosystem I can achieve an even higher energy level. It then passes two electrons to ferredoxin, which transfer a pair of electrons to NADP+ reductase. The role of NADP+ reductase is to convert NADP+ and

The proton gradient is utilized by the enzyme ATP synthase to create ATP from ADP and phosphate. Both ATP and NADPH will be utilized in the Calvin Cycle. The Calvin Cycle The Calvin Cycle is the process by which energy is transferred from short-term energy storage, ATP and NADPH, is used to fix carbon dioxide (CO2) into long-term energy storage molecules like glucose, sucrose, and starch. The process starts with the enzyme rubisco, catalyzing the fixation of three CO2 molecules and incorporating each into the sugar

Figure 4.

continued on next page www.carolina.com/distancelearning

5

EXPLORING PHOTOSYNTHESIS AND PLANT PIGMENTS Background continued ribulose bisphosphate (RuBP). The product of this reaction splits into two molecules of 3-phosphoglycerate (3-PGA), yielding a total of six 3-PGA molecules. Energy from six ATP and six NADPH are used in subsequent reactions to produce six glyceraldehyde-3-phosphate (G3P) molecules. One of these is used as a building block for glucose or other carbohydrates, and the other five are recycled, with the addition of three more ATP into three RuBP molecules, which can cycle through again. The ADP and NADP+ produced in the cycle are reused in the light reactions. Pigments and Chromatography Chromatography is a preferred analytical method for separating and identifying very small samples of a mixture. This analysis takes advantage of differences in the solubility and adsorption of substances by partitioning them between a mobile and a stationary phase. The mobile phase in this investigation is a liquid solvent, while the stationary phase is a solid (paper or silica gel). The chromatography activity will separate the pigments found in chloroplasts. Each pigment consists of a unique molecule whose properties give it a characteristic color. The paper chromatography has been designed to separate out different pigments if the sample contains more than one pigment. The pigments will dissolve in the solvent, isopropyl alcohol, and travel up the paper at different rates. Some will tend to bond more or less strongly to the cellulose in the paper and some are physically larger than others. These differences lead to the formation of different colored bands on the paper, one band for each pigment in the mixture. If the leaf contains only one pigment, expect

6

Carolina Distance Learning

to obtain only one color band. In the second activity, an investigation will be conducted that examines the rate of photosynthesis in various light conditions and CO2 concentrations. Fresh leaf disks will be prepared and then the oxygen and carbon dioxide will be removed from the leaves. The leaf disks will then be placed in light or dark environments with various amounts of CO2 in solution. The number of floating disks will be used to indicate photosynthetic activity.

Materials Included in the materials kit:

Needed from the equipment kit:

70% Isopropyl alcohol, 7 mL

2 Drinking straws

Chromatography paper strip

2 beakers, 250 mL

Plastic test Graduated cylinder, 25 mL tube

Baking soda

Medicine cup

Straight pin

Ruler

Test tube rack

4 Syringes, 10 mL

4 Syringe stands

Dropping Pipet

Grease pencil

Needed but not supplied: • Dish soap • Tap water • Coin (dime or quarter) • Light source (grow light or desk lamp) • Paper towels • Pencil • Plant leaves, 5–8 (fresh spinach recommended) • Timer or timing device • Scissors • Small bowl filled with tap water

Reorder Information: Replacement supplies for the Exploring Photosynthesis and Plant Pigments investigation (item number 580110) can be ordered from Carolina Biological Supply Company. Call: 800-334-5551 to order.

www.carolina.com/distancelearning

7

EXPLORING PHOTOSYNTHESIS AND PLANT PIGMENTS Safety

Preparation

Wear your goggles, gloves, and lab apron at all times while conducting this investigation.

1. Spinach leaves are recommended, but any kind of tender, dark green leaf will work. Refrigerate cut leaves until ready for use.

Read all the instructions for this laboratory activity before beginning. Follow the instructions closely and observe established laboratory safety practices, including the use of appropriate personal protective equipment (PPE) described in the Safety and Procedure sections. Isopropyl alcohol is highly flammable as both a liquid and vapor. Keep chemicals away from any heat or flame sources. Isopropyl alcohol can cause serious eye irritation and respiratory irritation. May cause drowsiness or dizziness. Do not eat, drink, or chew gum while performing this activity. Wash your hands with soap and water before and after performing the activity. Clean up the work area with soap and water after completing the investigation. Keep pets and children away from lab materials and equipment.

ACTIVITY 1 A Plant Pigments Overview In this activity, paper chromatography will be used to determine if a green leaf contains only one pigment or a mixture of pigments.

2. Clean and sanitize work area. 3. Gather needed materials. 4. Hold the chromatography paper on the edges to avoid getting oils on the wicking surface. Cut the piece of chromatography paper lengthwise into two, equal long strips. The extra strip is included should the experiment need to be repeated or the investigation is repeated with a second type of leaf.

Procedure 1. At 1.5 cm from the bottom of the chromatography paper strip, draw a faint pencil line across the strip. This line will be the origin line. Click here to view a video demonstrating how to transfer pigment to your chromatography strip: Paper Chromatography: Transferring Pigment to Paper http://players.brightcove. net/17907428001/HJ2y9UNi_default/ index.html?videoId=4578679011001 2. Lay a fresh spinach leaf on top of the origin line on the chromatography paper strip. 3. Roll the ribbed edge of the coin firmly on top of the leaf to press the pigments into the origin line.

continued on next page 8

Carolina Distance Learning

ACTIVITY

If the line is not present or is faint, repeat the previous step but make sure the green pigments are on the origin line. Reposition the leaf to ensure that the coin is rolling over fresh leaf tissue. 4. A vivid green stripe should be observed on the paper. 5. Place the plastic test tube in the test tube rack. 6. Add about 1 cm of isopropyl alcohol (approximately 20–25 drops) to the plastic test tube. The isopropyl alcohol will serve as the solvent that wicks and separates the plant pigments from the leaf up the paper. 7. Insert a straight pin into the center of each chromatography strip around 2 cm from the top. This will serve as a hanger for the strip when it is placed into the test tube. 8. Carefully lower the chromatography paper strip with the line oriented to the bottom, down the center of the test tube and into the solvent. The strip should be in the center of the tube, not touching the walls. Adjust the strip’s position by moving the pin side to side. The origin line with the pigments from the spinach must be above the liquid level yet the bottom of the paper must be in the liquid. 9. Allow the solvent front to wick up the chromatography paper and through the vivid green line until the solvent front reaches within a centimeter of the top. This should take around 10–15 minutes.

10. Remove the chromatography paper strip from the tube. 11. Remove the pin from the strip. 12. Place the strip on a paper towel, and mark the solvent front with a pencil before the solvent evaporates. Now is also a good time to take a photo of the results. 13. Outline each of the colored spots that contain the plant pigments using the pencil and number of them from bottom to top. 14. Measure the distance in millimeters that the solvent front traveled from the origin line to the solvent front. Record this value in Data Table 1. 15. Measure the distance in millimeters that each colored spot moved, from the origin to the middle of the spot. Record these values in Data Table 1.

Many factors can affect the rate at which solvents and pigments move through the chromatography paper. In order to compare results across different runs, scientists use a ratio, called a Retardation Factor (R f). The Rf is the ratio of the distance traveled by the center of a pigment spot to the distance traveled by the solvent front.

ACTIVITY 2 A Leaf Disk Assay Overview In this activity, an investigation will be conducted that examines the rate of photosynthesis in various light conditions and CO2 concentrations. Fresh leaf disks will be prepared and then the continued on next page www.carolina.com/distancelearning

9

ACTIVITY ACTIVITY 2 continued oxygen and carbon dioxide will be removed from the leaves. The leaf disks will then be placed in light or dark environments with various amounts of CO2 in solution. The number of floating disks will be used to indicate photosynthetic activity.

Preparation 1. Spinach leaves are recommended, but any kind of tender, dark green leaf will work. Refrigerate cut leaves until ready for use. 2. Clean and sanitize work area and gather all necessary materials. 3. Set up a light storage area. Create a storage place under bright light. Use a grow light or desk lamp, positioned over desk or table surface. At least a 60-watt equivalent bulb is recommended. 4. Set up a dark storage area. Create a dark storage space in a cleared cupboard, a dark room, or under a box. 5. Prepare soapy water by adding a drop of dish soap to about 20mL of water in the medicine cup and swirl to mix. 6. Add the contents of the baking soda bottle/ packet to a 250-mL beaker. 7. Carefully fill the beaker to 250 mL with distilled water. 8. Stir with a dropper pipet until the mixture is suspended. Not all of the powder will go into solution. 9. Quickly, before the suspended mixture has a chance to settle out, measure out 5 mL of the solution into a 25 mL graduated cylinder.

11. Fill the beaker to the 250-mL line. This is a 0.24% sodium bicarbonate solution. 12. Dispose of the contents of the first beaker. Be sure to rinse away all the residual bicarbonate. 13. Label the cleaned beaker 0.12%. 14. Place half of the 0.24% solution into the newly labeled beaker. Fill the 0.12% beaker to the 250-mL line. This is now a 0.12% solution. 15. Use the dropper pipet to transfer six drops of soapy water into the 0.24% beaker and 12 drops into the 0.12% beaker. 16. Label the 4 syringes “A”, “B”, “C”, and “D”.

Light Experiment 1. Cut out 40–50 leaf disks from the healthiestlooking leaves: a. Fill a small container with water. b. Lay the leaf on a hard surface, protected by a paper towel. c. Press the end of a plastic drinking straw into the leaf to cut a disk. d. Gently squeeze and twist on the end of the straw to remove the disks. Try not to damage the interior of the leaf disks. e. Keep the cut-out disks in the water container until you are ready to begin the experiment. It is important that the sizes of the disks are almost identical to ensure the surface area in each experimental group is the same.

10. Put the 5 mL in the second beaker. Label the second beaker 0.24%. continued on next page 10 Carolina Distance Learning

2. Click here to view a video demonstrating how to evacuate gas from the leaf disks: Extracting Gas from Leaf Disks http://players.brightcove. net/17907428001/HJ2y9UNi_default/ index.html?videoId=4886969626001 3. Gently place 10 leaf disks into each of the four syringes. Use the straw to gently position the disks close to the syringe tip 4. Replace the plunger and push towards the tip without smashing the disks. 5. Fill syringes “A” and “B” with solution from the “0.24%” beaker. a. Place the syringe tip into the liquid. b. Pull the plunger back to overfill the syringe to the 6-mL mark. c. Tap any bubbles out. d. Push the syringe tip firmly onto a syringe stand. Remove as many of the bubbles as possible from the solution in the syringe. Do this by tapping the sides of the syringe and expelling the solution and bubbles. Refill the syringe if necessary to get 6 mL of bubble-free solution. 6. Fill syringes “C” and “D” with solution from the “0.12%” beaker. a. Place the syringe tip into the liquid. b. Pull the plunger back to overfill the syringe to the 6-mL mark. c. Tap any bubbles out. d. Push the syringe tip firmly onto a syringe stand.

7. For each syringe, pull the plunger back gently until a slight vacuum pressure is felt. Do not pull the plunger past the 9 mL mark on the syringe. If the plunger comes This vacuum will pull any air from the spaces within the tissue of the leaf disks. Small air bubbles will form on the disks as a more negative pressure is created. out of the syringe, replace the leaves, refill the syringe with the appropriate solution, and begin again. 8. Gently shake the syringe or tap it on the side of the work surface while maintaining the vacuum. This should dislodge the air bubbles from the leaf disks. 9. Slowly release the plunger and allow the syringe to return to atmospheric pressure. Releasing the pressure suddenly may cause the cap to pop off. 10. Remove the cap and push out any gas that may have accumulated in the tip. 11. Continue the cycle of applying pressure and rem...