A.  Learning Objectives

In this lab, students will:

• use a spectroscope to observe the component wavelengths of white light.

• determine which wavelengths are absorbed or transmitted by filters and plant

pigments.

• determine under which conditions a plant leaf produces starch or utilizes starch stores.

• determine which pigments are most efficient in supporting photosynthesis

 

B. Textbook Correlation: 

Please review Chapter 8: Photosynthesis when preparing for the lab.

 

C.  Introduction

Write a three paragraph introduction to photsysnthesis.

 

     Autotrophs are organisms that synthesize food using the molecules and substances available in their environment. Photoautotrophs are a type of autotroph which use light energy to create food. Autotrophs are known as producers, because they produce their own food which can then be used by heterotrophs. Heterotrophs are organisms that cannot synthesize their own food, and must rely on the consumption of other organisms to get nutrients. These organisms are called consumers. Examples of organisms that are autotrophs are green plants, algae, trees, or anything else that relies on photosynthesis. Examples of heterotrophs include all animal and fungal species, as well as most bacteria.

 

In paragraph 2, summarize the process of photosynthesis.  Include the major chemical reaction (reactants and products) and the change in free energy.  Discuss the different wavelengths of visible light, photons, and the different quantity of energy associated with the individual wavelengths of light.  Include an images/animations about the individual wavelengths of visible light.

 

The chemical reaction of photosynthesis is 6CO2 + 6H2O = C6H12O6 + 6O2 + 6H2O. The reactants are carbon dioxide and water, while the products are glucose, oxygen, and water. The delta G of the whole reaction is +686 because cellular respiration is -686 and photosynthesis is the reverse of cellular respiration. Photosynthesis is driven by light energy. There are many different wavelengths of light energy that contains various amounts of energy. As seen in the diagram below of the electromagnetic spectrum, the visible light energy has many different wavelengths. The shorter the wavelengths, the more energy it contains. For instance, the purple area of the diagram has about a 400 nanometers wavelength, which means that it has more energy than the red area with about a 700 nanometer wavelength. Photons are small particles of light that travels in wavelike patterns and  and moves at the speed of light. 

 

Figure legend for wavelengths of light.

 

In paragraph 3, summarize the light-dependent and light-independent reactions of photosynthesis.  In your summary, include cellular location, discuss the role of pigments and excitable electrons in the reaction.  Also, discuss the energy intermediates that link the two processes and how a delay in either the light-dependent or the light-independent reactions affects the other set of reactions.  Include a useful image/animation for both the light-dependent and light-independent reactions.

Light reactions are a series of steps that lead to the production of ATP and NADPH that will then be fed in to the light-dependent reactions in order to create glucose. The light reactions take place within the thylakoid membrane of the chloroplasts. Its main components are photosystems II and I, NADP+ reductase, and ATP synthase. It starts with light of 680nm hitting the pigments molecules in PS II, causing them to absorb the energy  and become excited and they transfer it to the other pigments through resonance transfers. Eventually the energy reaches the reaction center where an electron is transferred to the primary electron acceptor. This causes PS II to lose electrons which are regenerated from the removal of electrons from water. The electrons then move down the ETC to PS I where it is hit with 700nm light. Then the energy moves down the ETC to NADP+ reductase in order to reduce NADP+ to NADPH. These reactions also create a H+ concentration gradient which powers ATP synthase also creating ATP. This is noncyclic flow which focuses on creation of NADPH. In cyclic, the energy never gets to NADP+ reductase, it cycles between Qb and PS I creating more of a H+ gradient which leads to more ATP.

The ATP and NADPH created from the light reactions are then fed in to the dark reactions within the stroma. The Dark Cycle (Calvin Cycle) is composed of steps in order to use ATP and NADPH to synthesize carbohydrates. Its composed of 3 major steps: carbon fixation, reduction and carbohydrate production, and regeneration of ribulose.  Carbon is first added to CO2 to create 3-PGs (3-phosphoglycerate) which is then converted to 1,3-BPG (biphosphoglycerate) by ATP. Then NADPH is used to donate high energy electrons to reduce 1,3-BPG to G3P that can be used in cellular respiration. The rest of the G3P that is left is converted back to RuBP in order to continue the cycle by ATP. This ends up with glucose and NADP+ and ADP.

A delay in either the light or dark reactions affects the other set of reactions because each reaction creates a substance that is needed to continue the other reaction. If there is a delay in the light reaction, there might not be enough ATP or NADPH to continue the dark reactions. If there is a delay in the dark reaction, there might not be enough NADP+ to create more NADPH. So the cell has to time the reactions so that the cell can work at a maximum efficiency.

 

INSERT IMAGE/ANIMATION HERE
Figure legend for the light-dependent reactions	Figure legend for the light-independent reactions

     

In the following series of exercises, you will learn how to use a spectroscope to view the different wavelengths composing white light, investigate how leaves kept in light and dark store and utilize glucose, study chloroplast structure, and investigate how Elodea leaves fix carbon.

 

D.  Light Absorption by Photosynthetic Pigments

Colored substances can either absorb particular wavelengths of light or reflect them. Wavelengths that are absorbed are taken up by the colored substance and therefore are not available for our eyes to detect them. On the other hand, wavelengths that are reflected by a colored surface are the ones that can enter our eyes and strike the retina. These are the colors that we see.  A filter absorbs a particular wavelength of light and transmits (allows to pass through) the rest.  This is important because plants must contain pigment molecules capable of absorbing light waves in order to use the energy they contain for photosynthesis.

 

In this exercise you will use a spectroscope to observe the component wavelengths of white light and then determine which wavelengths are absorbed or transmitted by filters and plant pigments such as chlorophyll.

 

Procedure

1. A spectroscope uses a prism to separate white light into its component wavelengths. This spectrum can be observed along with a scale indicating the wavelength associated with each color.

 

2. Use colored pencils to construct the spectrum of white light you observe using the spectroscope.

 

 

 

 

 

3. Now place a red filter over the spectroscope slit.

 

Which colors do you observe in the spectroscope? ______________________________

 

Which colors were absorbed by the filter? _____________________________________

 

4. Repeat using the blue and green filter.

 

Colors observed with the blue filter. _________________________________________

 

Colors absorbed by the blue filter. ___________________________________________

 

Colors observed with the green filter. ________________________________________

 

Colors absorbed by the green filter. __________________________________________

 

5. Now hold a test tube of plant pigment extract in front of the spectroscope. Some of the light waves will be reflected, giving the extract the color you perceive. Other wavelengths will be absorbed by the pigments. These are the wavelengths that will pass into the spectroscope where you can observe them. These are also the wavelengths available to the plant cells for photosynthesis.

 

6. Observe the spectrum in the spectroscope and record the colors and wavelengths you observe.

 

7. What difference do you note between the spectra produced by a green filter and by the plant extract?   Is green really the color of photosynthesis as is commonly characterized?  If not, explain your answer.   

 

E.  Requirement of Light for Starch Production

As noted in the introduction, plants produce glucose, using some immediately for cellular respiration and storing the rest as starch. This exercise will  measure the requirement for light energy in the production of starch.  We will work with Germanium leaves that have a small section covered so that light can not strike it. In this exercise you will remove all the pigments from these leaves and test each for the presence of starch.

 

Predictions

Before conducting this exercise, test your understanding of photosynthesis by predicting if starch will be present:

a) in an uncovered light-grown leaf

b) in a covered light-grown leaf

c) in an uncovered dark-grown leaf

d) in a covered dark-grown leaf

 

Procedure

1. Place the water beaker on the hot plate and bring to a boil.

2. After the water has boiled, place 150 mL of 95% ethanol onto the hot plate. The boiling point for ethanol if much lower than that of water, so it will come to a boil quickly.

3. Take the light-grown leaf, remove its covering and place it in the boiling water for one minute. This kills the plant cells and breaks open its membranes.

4. Remove the leaf from the boiling water using the tweezers and place it in the boiling ethanol. This removes the pigments from the leaf. Leave it in the ethanol until the leaf turns white.

5. Remove the leaf from the ethanol and return it to it’s the petri dish filled with water.

6.  Place the leaf into the pertri dish with iodine (Lugol’s solution).  This stain reacts with starch to produce an intense dark blue-black color. Allow the leaves to soak in the Lugol’s solution for 2-3 minutes.

7.  Return the leaf to the water dish. Use the tweezers to spread it out flat.

8.  Observe the staining pattern for the leaf. Draw what you observe in Figure 4 and answer the following questions.

 

 

 

 

 

   

Questions

1. When a section of the leaf stains blue-black, what does that mean?

 

 

2. When a section of the leaf fails to stain blue-black, what does that mean?

 

 

3. Is there a difference in the staining of the covered vs. uncovered sections? Explain why.

 

 

 

F.  Measuring the Efficiency of Different Pigments in Absorbing Light
Pigments of a leaf act as solar panels.  They absorb sunlight and convert that energy to the chemical energy of ATP and NADPH.  Some pigments are more efficient than others in supporting photosynthesis.   The main photosynthetic pigment is called chlorophyll and provides the green color observed in many leaves.  There are other leaf pigments, like carotenoids (yellow) and anthocyanin (pink) found in the leaves of the ornamental Coleus plant.  The goal of this experiment is to determine the efficiency of the different pigments in supporting the process of photosynthesis.

 

Procedure:

1.  Remove a leaf from the Coleus plant.  Sketch the distribution of colors within the leaf and note the pigment responsible.

 

 

        

                  Coleus leaf with intact pigment                                       Coleus leaf after staining with iodine

 

2. Place the water beaker on the hot plate and bring to a boil.

3. After the water has boiled, place 150 mL of 95% ethanol onto the hot plate. The boiling point for ethanol if much lower than that of water, so it will come to a boil quickly.

4. Take the Coleus leaf, remove its covering and place it in the boiling water for one minute. This kills the plant cells and breaks open its membranes.

5.  Remove the leaf from the boiling water using the tweezers and place it in the boiling ethanol. This removes the pigments from the leaf. Leave it in the ethanol until the leaf turns white.

6.   Remove the leaf from the ethanol and return it to it’s the petri dish filled with water.

7.  Place the leaf into the pertri dish with iodine (Lugol’s solution).  This stain reacts with starch to produce an intense dark blue-black color. Allow the leaves to soak in the Lugol’s solution for 2-3 minutes.

8.  Return the leaf to its dish the water dish. Use the tweezers to spread it out flat.

9.  Observe the staining pattern of each for the leaf. Draw what you observe and answer the following questions.

Questions:

1.  Which pigment was most efficient in supporting photosynthesis?  Explain.

 

 

 

2.  Which pigment was least efficient in supporting photosynthesis?  Explain.

 

 

 

   

G.  Post-Lab Questions

1. In order for photosynthesis to occur the following must be present (hint: review the photosynthesis equation):

a. ________________________________ as the energy source.

b. ________________________________ as the carbon source.

c. ________________________________ as the electron donor.

d. ________________________________ for the absorption of light energy.

 

2. Explain why grow lights for house plants are never green.

 

    

 

3. Explain why plants that have been kept in the dark for a week will still be able to conduct cellular respiration.

 

 

 

 

 

4. As you saw from your chromatogram, there are more pigments present in leaves than you can see. Propose an explanation for why leaves only appear green in the growing season, but turn bright colors in the autumn when the weather turns cold.