LAB TOPIC 7  

Photosynthesis: Capture of Light Energy

Adapted from Perry, Morton and Perry, Laboratory Manual for General Biology (8^th Edition) and Mader, Laboratory Manual:  Biology (9^th edition)

Laboratory Objectives: 

After completing this exercise, you will be able to

1. Define photosynthesis, autotroph, heterotroph, chlorophyll, chromatogram, absorption spectrum, carotenoid;

2. Describe the role of carbon dioxide in photosynthesis;
3. Describe how white light is composed of many colors of light.
4. Explain an experiment that indicates that white light promotes photosynthesis.
5. Determine the effect of light and carbon dioxide on photosynthesis; 6. Explain the effectiveness of various colors of light for photosynthesis. 

7. Determine the wavelengths absorbed by pigments;
8. Describe the separation of plant pigments by paper chromatography, and identify the pigments in spinach chloroplast extract;
9. Describe an experiment that indicates carbon dioxide is utilized during photosynthesis. 
10. Identify the carbohydrate produced in geranium leaves during photosynthesis;
11. Describe the relationship between cellular respiration and photosynthesis. 
12. Identify the structures composing the chloroplast and indicate the function of each structure in photosynthesis.

 

 

Introduction

Photosynthesis is the process by which light energy converts inorganic compounds to organic molecules and the by-product, oxygen. It may be the most important biological event. Without it, most living things would starve, and atmospheric oxygen would become so low, the planet could no longer support animal life. Ultimately, the source of light energy is the sun, although on a small scale we can substitute artificial light. Nutritionally, two types of organisms exist in our world, autotrophs and heterotrophs. Autotrophs (auto means self, troph means feeding) synthesize organic molecules (carbohydrates) from inorganic carbon dioxide. The vast majority of autotrophs are the photosynthetic organisms that you're familiar with -- plants, as well as some protistans and bacteria. These organisms use light energy to produce carbohydrates. (A few bacteria produce their organic carbon compounds chemosynthetically, that is, using chemical energy.)

By contrast, heterotrophs must rely directly or indirectly on autotrophs for their nutritional carbon and metabolic energy. Heterotrophs include animals, fungi, many protistans, and most bacteria.

In both autotrophs and heterotrophs, carbohydrates originally produced by photosynthesis are broken down by cellular respiration (Lab Topic 7), releasing the energy captured from the sun and transferring it to other molecules to be available  for metabolic needs.

The photosynthetic reaction is often conveniently summarized by the equation:

 

12H₂O  +  6CO₂        light energy         6O₂  +   C₆H₁₂O₆  +   6H₂O               water carbon        -------------à        oxygen       glucose          water                             dioxide

 

The following experiments will acquaint you with the principles of photosynthesis. At the end of the laboratory, be sure you understand each one.  

 

Exercise 7.1 : Test for Starch 

Although glucose is often produced during photosynthesis, it is usually converted to another compound for transport or storage. In plants and many protistans, the most common storage carbohydrate is starch, a compound made up of numerous glucose units linked together. Starch is designated by the chemical formula (CH₂O)_n where n indicates a large number. Most plants transport carbohydrate as sucrose.  Remember that you learned to test for starch with iodine in Week 4.  What does a positive test for starch look like?_____________________________________________________a)  

 

 

Exercise 7.4 : Necessity of Photosynthetic Pigments for Photosynthesis

Coleus plants are widely planted ornamentals that are popular for their striking foliage color patterns. Observe the plants available in the lab and note their wide variety and attractiveness. This experiment addresses the hypothesis that chlorophyll is necessary for photosynthesis to occur.

MATERIALS

Per student group (4):           Per lab room:

• colored pencils or pens	• variegated Coleus plants
• two 400-mL beakers	• source of dH2O
• hot plate in fume hood
• heat-resistant glove
• petri dish halves
• bottle of iodine solution
• bottle of 95% ethanol (EtOH)
• forceps

 

Procedure

1. Obtain a leaf of variegated Coleus. In the left-hand circle of Figure 7.3 at the end of the lab topic, carefully sketch the leaf, indicating the distribution of each color on the leaf with colored pencils or pens. Green coloration is due to chlorophyll, the major photosynthetic pigment. Pink colors are caused by water-soluble anthocyanin pigments (not involved in photosynthesis) (remember our beet experiments?), and yellows are formed by carotenoid pigments. Be sure that you look at both surfaces of the leaf in case pigment distribution differs.

Make a prediction regarding starch presence and photosynthetic activity for each

pigmentation area:                                                                                                    i)

 

 

2. As above, kill and extract the pigments from the leaf: 
   1. With a china marker, label the beakers A (for alcohol) and dH₂O. 
   2. Add about 150 mL distilled water to the dH₂O beaker, set it on the hot plate, and turn on the hot plate to the highest setting. Allow the water to come to a boil. 
   3. Alcohol has a much lower boiling point than water, and so takes very little time to come to a boil. When the water is boiling, put about 150 mL of alcohol in the A beaker, set it also on the hot plate, and bring to a boil. Keep the alcohol beaker covered with aluminum foil as much as possible throughout the lab to prevent excess evaporation.     (d) Place the leaf from one treatment in the beaker of boiling water for about 1 minute. This kills the tissue and breaks down internal membranes.

1. 5. Use the long forceps to move the wilted leaf from the water into the boiling alcohol. This will extract the photosynthetic pigments from the plant tissues. When the pigments have been extracted, the liquid will appear green, and the leaf will appear to be mostly bleached.
   6. Remove the leaf from the alcohol with forceps, and dip momentarily in the boiling water to soften.

 

3. Test the leaf for the presence and localization of starch:
   1. Place the killed, depigmented leaf in a petri dish filled with iodine solution.           (b) Let the leaf soak in the iodine solution for a couple minutes, rinse, and float in water in another petri dish in order to observe the pattern of staining.

 

4. On the right-hand side of Figure 7.3 at the end of the Lab Topic, resketch the leaf, indicating the pattern of staining with iodine.

 

5. How does the pattern of starch storage relate to the distribution of chlorophyll?   j)
6. Write a conclusion either accepting or rejecting the hypothesis.                           k)

 

 

Exercise 7.5 : Absorption of Light by Chloroplast Extract

White (sun) light contains different colors of light, as demonstrated when white light passes through a prism (see Figure 7.4, and your instructor’s demonstration).  

 

 

 

Figure 7.4  White light.  White light is made up of various colors, as can be seen when white light passes through a prism. (Mader, Figure 6.3)

 

 

 

 

White light is best for photosynthesis because it contains all the colors of

light.  However, the pigments present in plants abosrb some colors of light better than others. Use Figure 7.4, the colored overhead projection and the experiment below to learn about the absorption of plant pigments.

 

MATERIALS

Per student group (4):           Per lab room:

• colored pencils (red, violet, blue, yellow, green, orange)	• prism
• spectrophotometer	• overhead projection of wavelengths and light colors
• tube containing petroleum ether blank	• continuous spectrum scan of plant extract
• tube containing plant extract in	• graph paper
petroleum ether

 The technician has prepared an extract from spinach leaves and a sample of it, dissolved in petroleum ether, can be found at each spectrophotometer along with a sample of petroleum ether alone. The extract and the pure petroleum ether are in special optical tubes known as cuvettes. You will work in groups of four or whatever number the instructor designates. 

1. Turn on the power and give it a few minutes to warm up. Set the wavelength at 700 nm and make sure that the filter (on the front edge) is correct for that wavelength.  Don’t forget to check that the proper filter is in place at EACH wavelength.  
2. Set the mode to Transmittance.
3. Zero the machine with the left knob
4. Change the mode to Absorbance (push the button).
5. Insert the pet ether cuvette (called the blank) into the chamber. Adjust the instrument to zero absorbance with the right side knob.   
6. Remove the cuvette
7. Place the chlorophyll sample in the spectrophotometer, close the lid and read the absorbance directly from the meter.  Record your result (See Table 3 at end of Lab Topic). 
8. Set the instrument at 680nm and repeat the above steps (wavelength, check filter, blank, then sample). Continue doing this at 20nm decrements (keep lowering the wavelength by 20nm) until you reach 400nm.
9. Using the graph paper provided,or using the computer, make a graph of the absorption spectrum of the chloroplast extract.  On the X axis, list the wavelengths from 700nm to 400nm. 
10. Using colored pencils and the overhead projected wavelength figure, make some indication as to the color of each wavelength or range of wavelengths. 
11. On the Y axis indicate the absorbance from 0A to 2A.

 The absorption spectrum on the overhead projection is smoother because it is prepared using a spectrophotometer that is much more sophisticated than the instrument you used. This spectrophotometer is a double beam instrument that automatically subtracts the blank while scanning the desired range of wavelengths and continuously recording the results on a moving graph sheet. Compare your absorption spectrum with the ovrhead.  

 

What wavelengths of light (colors) are absorbed most by the extract?                      l)

What wavelengths of light (colors) are absorbed least?                                           m) Does the color that is absorbed least make any sense considering your every day

observation of plants? Explain.                                                                               n)

 

Exercise 7.6 : Separation of Photosynthetic Pigments by Paper Chromatography Paper chromatography allows substances to be separated from one another on the basis of their physical characteristics. You will apply a mixture of the pigments present in leaves by rubbing them on the chromotography paper. You will use paper chromatography to separate any pigments present.  Separation occurs due to the solubility of the pigment in the chromatography solvent and the affinity of the pigments for absorp-tion to the paper surface. The finished product, showing separated pigments, is called a chromatogram.

 

MATERIALS

Per student group:          

• chromatography paper,        3 cm x 15 cm strip	• chromatography chamber  (large test tube)
• small metric ruler	• solvent (10% acetone in petroleum ether)
• green leaf	• cork with paper clip
• scissors	colored pencils (green, blue-green, yellow, orange)

 

Procedure

1. Obtain a 3 cm x 15 cm sheet of chromatography paper. Touch only the edges of the paper, because oil from your fingers can interfere with development of the chromatogram. Cut the end to form a point. Mark a dot with a pencil about 3 cm from the point of the paper.
2. Using the handle of the scissors, rub the leaf several times to make a strong narrow green stripe across the paper strip where you put the dot, about 3 cm from the point of the paper.
3. Allow the pigment stripe to dry. 

Caution:  Avoid inhaling the solvent vapors.  Keep the chambers tightly capped whenever possible.   Dispose of the solvent in the waste jar at the front of the classroom, NOT down the sink.

4. Pour solvent into the test tube until it reaches 2 cms deep.  Adjust the length of the paper so that the tip will just rest in the solvent, attach it to the cork clip, and insert it into the test tube.  Be sure that the sample stripe is above the solvent, that the paper is hanging without a curve in it, and that the cork is on tightly.  

 

5. Watch the separation take place over the next 10 minutes. When the solvent is within about 1 cm of the top of the paper, the separation is complete. Remove the strip, close the chromatography chamber, and allow the chromatogram to dry. Properly dispose of the solvent.  
6. Using colored pencils, sketch your results in Figure 7.4, showing the relative position of the colors along the paper.

 

 

 

 

7. Beginning nearest the original pigment spot, identify and label the yellow-green pigment chlorophyll b.  Moving upward, find the blue-green chlorophyll a, two yelloworange xanthophylls in the middle, and an orange carotene at the top. Xanthophylls and carotenes belong to the class of pigments called carotenoids.

9.  You may preserve your chromatogram for future reference by keeping it in a dark place (for example, between the pages of your textbook). Light causes the chromatogram to fade.

 

What pigments are contained within the chloroplasts of spinach leaves?            o) What common "vegetable" is particularly high in carotenes?                              p) Exercise 7.7 : Fluorescence Theodore Marcus, Ed.D.

 

You are probably aware of the fact that light energy is the driving force that is responsible for the process of photosynthesis. The experiments that you have conducted during this laboratory exercise are designed to give you further insight into the specific role that light energy plays in this vital process.

 

Under normal conditions the electrons of an atom are in an unexcited or ground state, referring to the fact that the electrons remain at their lowest energy levels. When an electron in an atom absorbs energy, it rises to a higher orbital level. An electron occupying a higher than normal orbital level possesses a greater amount of energy and is referred to as being in an excited state.

 

In order to be in an "excited" state, a specific amount of energy has to be absorbed by an electron. In photosynthesis. the energy absorbed by the electrons comes from light. The light energy is provided in the form of photons. A photon is generally regarded as a discrete particle of light energy, having zero mass, no electrical charge and an indefinitely long lifetime. When a photon strikes a chlorophyll molecule, the energy of the photon is transferred to an electron in the chlorophyll molecule, thus raising the energy level of the electron. This excited electron now has the potential to pass its energy to other atoms.

 

In an alcoholic solution of chlorophyll, the normally present electron acceptors of the excited electrons are either absent or have been altered in the boiling process by which the chlorophyll has been obtained and thus the excited electrons cannot be transferred to other atoms. As a result, the excited electrons fall back down to their ground or unexcited state. In so doing, the excited electrons give up the energy that they absorbed from the photons. The energy is released in the form of light that is emitted in the visible red spectrum. The light energy that is emitted by the falling electrons is referred to as

FLUORESCENCE.

 

A simple experiment can be performed that will demonstrate the ability of an alcoholic suspension of chlorophyll to fluoresce. For best results, the following procedure should be conducted in a dimly lit or darkened room.

 

1. Fill a standard test tube 3/4 full with an alcoholic suspension of chlorophyll.

 

2. Examine the test tube containing the chlorophyll by both transmitted and reflected light. If available, utilize the narrow beam of light produced by laser pointers.
3. Under which of the two light conditions (transmitted or reflected) is the chlorophyll exhibiting the property of fluorescence?              q)
4. What other color do you observe? Offer an explanation for the appearance of this color.     r)

 

 

Pre-Lab Questions.  Photosynthesis. Circle the correct answer

1. The raw materials used for  photosynthesis include             
   1. O₂            
   2. C₆H₁₂O₆             
   3. CO₂  + H₂O                 
   4. CH₂O                
2. A device useful for viewing the spectrum of light is a            
   1. spectroscope                
   2. volumeter                             (c) chromatogram   

          (d) chloroplast                   

3. Products and byproducts of photosynthesis do NOT include
   1. O₂            
   2. C₆H₁₂O₆                      (c) CO₂

          (d) H₂O                   

4. A paper chromatogram is useful for     (a) measuring the amount of      photosynthesis

    (b) determining the amount of gas      evolved during photosynthesis     (c) separating pigments based on their       physical characteristics     

    (d) determining the distribution of        chlorophyll in a leaf     5.  Which of the following pigments would you find in a geranium leaf? 

1. 1. chlorophyll, xanthophyll,    phycobilins          
   2. chlorophyll a, chlorophyll b,      carotenoids    (c) phycocyanin, xanthophyll,    fucoxanthin

    (d) carotenoids, chlorophylls,       phycoerythrin            

 

 

                     

6.  Which reagent would you use to determine the distribution of the carbohydrate stored in leaves?  (a) starch 

2. Benedict's solution
3. chlorophyll 
4. I₂KI 

7.  An example of a heterotrophic organism is    (a) a plant 

2. a geranium 
3. a human 
4. none of the above  8.  Organisms capable of producing their own food are known as 

1. autotrophs 
2. heterotrophs 
3. omnnivores 
4. herbivores 

9. Grana are                                

   (a) the same as starch grains    (b) the site of ATP production within    chloroplasts

9. 3. part of the outer chloroplast    membrane
   4. contained within mitochondria        and  nuclei
10. The ultimate source of energy trapped during photosynthesis is
    1. CO₂
    2. H₂O        
    3. O₂
    4. sunlight

           

 

Answer Sheet Photosynthesis Exercises Exercise  7.1 a)

 

Exercise  7.2

TABLE 7-1 Effect of Light and Carbon Dioxide on Starch Presence

Plant Growing Condition	Appearance	Prediction	Results: Starch Presence and Location
I. Normal conditions with both light and carbon dioxide
II.  In dark, with normal carbon dioxide
III.  In light, but with carbon dioxide removed

Figure 7.1 Distribution of starch in Plants. (a) Normal light and CO₂; (b) dark, normal CO₂; (c) light, no CO₂.

 

 

b)

 

c)

 

d)

 

e)

 

 

 

Exercise 7.3

 

TABLE 7.2 Relationship Between Light and Starch Production

 

                                                 Prediction   Starch Presence and Location

 

Geranium Plant
Growing Condition	Masked Areas	Unmasked Areas	Masked Areas	Unmasked Areas
Light-grown
Dark-grown

.  

 

 

Figure 7.2 Distribution of the photosynthetic product _______________.

 

f)

 

g)

 

h)

 

 

 

 

Exercise 7.4

i)  

 

          

 

 

Figure 7.3.  Left:  Coleus leaf before staining.  Right:  Coleus leaf after staining. 

 

j)

 

 

k)

 

7.5

 

Table 7.3  Absorption of Light at Various Wavelengths

700		600		500
680		580		480
660		560		460
640		540		440
620		520		420
				400

 

l)

 

m)

 

n)

 

7.6  

Draw your chromatography results.

 

 

 

Figure 7.4 Chloroplast pigment chromatogram.  Labels:   chlorophyll b, chlorophyll a, 

xanthophylls, carotene

 

 

 

 

 

 

 

 

 

o)

 

p)

 

 

Exercise 7.7 : Fluorescence

 

q)

 

 

r)

 

 

 

 

 

 

 

 

 

 

 

7.8 : Chloroplast Structure

 

 

Figure 7.5 left. Photosynthesis includes the light reactions when energy is collected and oxygen is realeased, and the Calvin cycle reactions when carbohydrate (CH₂O) is formed.  Figure 7.5 right. The arrangement of membranes and compartments inside a chloroplast.

 

The chloroplast is the organelle concerned with photosynthesis. Photosynthesis includes the light reactions when energy is collected and oxygen is realeased, and the Calvin cycle reactions when carbohydrate (CH₂O) is formed (Figure 7.5 left).

 

Study Figure 7.5 (right), an artist's conception of the three-dimensional structure of a chloroplast.  Like the mitochondrion and the nucleus, the chloroplast is surrounded by two membranes. Within the stroma (semifluid matrix), identify the thylakoid disks stacked into grana (a single stack is a granum). The chloroplast pigment molecules are located on the surface of the thylakoid disks. Hydrogen ion buildup occurs within the interior of the disks. As these ions are expelled back into the stroma, ATP is formed. Within the stroma, the ATP is used to generate organic compounds. These compounds are converted to carbohydrates, lipids, and amino acids from carbon dioxide, water, and other raw materials.

 

Now examine Figure 7.6, below, a high-magnification electron micrograph of a chloroplast. With the aid of Figure 7.5, label the micrograph.

If the plant is killed and fixed for electron microscopy after being exposed to strong light, the chloroplasts will contain starch grains. Note the large starch grain present in this chloroplast. (Starch grains appear as ellipsoidal white structures in  electron micrographs.)

 

 

 

     Figure 7.6.  Electron micrograph of chloroplast (10,000X).  Inset:  a single granum        (20,000X).  Labels:  chloroplast membrane, thylakoid disks, stroma , starch. 

 

Lab Topic 7 : Photosynthesis: Capture of Light Energy

 

POST-LAB QUESTIONS  EXPERIMENTAL REVIEW

 

Experiment. Effects of Light and Carbon on Starch Production

1. Is starch stored in the leaves of some plants? Would you expect leaves in a temperate climate plant to be the primary area for long-term starch storage? Why or why not? What part(s) of a plant might be better-suited for long-term starch storage?

 

 

 

 

 

Experiment. Relationship Between Light and Photosynthetic Products

2. Examine the photo at right, which shows the location of starch in two geranium leaves treated in much the same way as you did in Exercise 7.3.

Explain the results you see.