Laboratory Notes for BIO 1003

© 30 August 1999, John H. Wahlert, Mary Jean Holland, & Joan Japha


©15 June 2000, Joan Japha


Heterotrophic organisms such as fungi and bacteria obtain the energy they need for growth, reproduction, and movement by decomposing other organisms or molecules. Members of the Kingdom Plantae, together with some members of the Kingdom Protista and all of the cyanobacteria (Domain Bacteria), are photosynthetic organisms; as such, they are autotrophs: they synthesize their own food by using simple raw materials plus the energy of sunlight. They are responsible for renewing the energy supplies available on the Earth. Coal, gasoline, and heating oil fuel our society. These sources of energy are largely derived from the metabolic activity of plants that lived millions of years ago. Members of the Kingdom Animalia, heterotrophic organisms including ourselves, obtain energy from the food they eat. In addition, the process of photosynthesis is the source of oxygen required for the respiration of both plants and animals.

The process of photosynthesis transduces (converts) the kinetic energy of sunlight into the potential energy of chemical bonds. The energy is initially trapped in ATP molecules, later incorporated into the bonds of glucose, and eventually stored as carbohydrates—sugar or starch. Because this laboaratory exercise is about the Kingdom Plantae, the process of photosynthesis will be examined as it is carried out in the chloroplasts of plant cells.

The process of photosynthesis is a complex series of chemical reactions that begins with carbon dioxide and water (molecules of low potential energy) and ends with carbohydrates such as glucose and starch (molecules of high potential energy). The metabolic activity of plants enables the radiant energy of sunlight to be transduced (converted) to the energy found in the chemical bonds of carbohydrates.

Chlorophyll, the photosynthetic pigment in chloroplasts, absorbs light energy. Plants appear green because chlorophyll does not absorb light in the yellow-green region of the visible spectrum. Yellow and green wavelengths of the spectrum are reflected or transmitted depending on the thickness of a leaf. Chlorophyll absorbs light in the blue and red regions of the spectrum. The energy absorbed by chlorophyll raises the energy level of electrons in the active center of the chlorophyll molecule. In the chloroplast, the movement of these high-energy electrons is coordinated with the formation of the high-energy molecule, ATP, and the electron carrier, NADPH. During this process, water molecules are split and oxygen gas is released. This is origin of the oxygen in the air we breathe.

The importance of photosynthesis may be appreciated by considering the overall reaction:

6CO2    +    6H2O    +    light    [arrow]    C6H12O6    +    6O2
water energy glucose
(food we eat)
(gas in the air
we breathe)

The overall process of photosynthesis has two parts, each consisting of groups of reactions with different purposes. The fluid inside a chloroplast is called stroma; the membranes inside the chloroplast in which the chlorophyll molecules are embedded are called thylakoids; in many places the thylakoids are closely stacked and appear as dark green dots or grana (grains) in each chloroplast.

1. The Light-Dependent Reactions require the presence of light. In green plants, these reactions take place within the chloroplast, and the enzymes that catalyze these reactions are integral parts of the thylakoid membranes, the membranes found within the chloroplast. As a result of the Light-Dependent Reactions, light energy is transduced to the chemical energy in the bonds of ATP. NADP+ is reduced when it picks up a pair of electrons to become NADPH. Water molecules are oxidized during the Light-Dependent Reactions and oxygen gas is released.

ADP    +    phosphate group    +    light energy [arrow] ATP

NADP+    +    electrons    +    hydrogen ion    +    light energy [arrow] NADPH

2. ATP and NADPH enter the Light-Independent Reactions. These reactions take place in the stroma of the chloroplast. This is the part of the interior of the chloroplast that is not occupied by the thylakoid membranes. During the Light-Independent Reactions, the energy associated with ATP and the hydrogen and electrons carried by NADPH are incorporated into a carbohydrate. This process is also called carbon dioxide fixation, meaning that carbon dioxide from the air is bound (fixed) into a stable carbohydrate. The simple carbohydrate that is formed may then be converted to glucose or to starch for storage. These reactions can be outlined as:

a.      CO2    +    NADPH    +    ATP [arrow] G-3P
  glyceraldehyde-3 phosphate
(simple carbohydrate)

b.      simple carbohydrate [arrow] glucose [arrow] starch



The goals of the laboratory exercises are:

  1. to extract the photosynthetic pigment from the leaf and study some of its properties;
  2. to test for photosynthetic products in a leaf.

A. Before extracting chlorophyll from the cells of a Coleus leaf, note that the leaves contain other pigments in addition to chlorophyll. Observe the leaf and sketch its pigment pattern.

  1. Boil some leaves in a small amount of water in a beaker on the hot plate. Does the water change color? What has happened?

  2. Carefully pour off (decant) the hot water from the beaker and cover the leaf with alcohol. Boil the leaf gently (Why should the heat be set lower than for water?) Does the alcohol become green? What has happened? What does this indicate about the nature of chlorophyll? What has happened to the red pigment?

  3. Carefully pour the colored alcohol solution into a test tube and save the leaf for Part 3.

  4. Use the spectroscope to examine the spectrum of the chlorophyll. Examine the complete spectrum of white light in the spectroscope and note that it is broadly divided into a red band, a yellow-green, and a blue band. Now place a tube containing a fairly concentrated solution of chlorophyll between the spectroscope slit and the lamp. (If it is concentrated enough, use the solution saved in PART B above.) Which light bands are strongly absorbed? Which are transmitted most? What does this indicate about the function of chlorophyll? Would plants grow indoors using only a green light source?

B. What happens to the radiant energy absorbed by the chlorophyll in the solution? Fluorescence occurs when molecules absorb light of one wavelength and then emit light of a longer wavelength; this property can be observed in chlorophyll.

  1. Place a solution of chlorophyll in a beaker and shine a narrow beam of light through the beaker. Look along the path of the beam of light within the solution. What is the color of the solution along the path of the beam of light? (Darken the room if necessary to ensure that the path of the beam of light is visible in the solution.)

  2. In the absence of the organized membranes of the chloroplast, light absorbed by chlorophyll molecules in solution is given up and emitted as light of a longer wavelength than that originally absorbed. Look into the beaker and notice that red light (long wavelengths) is visible along the path of the light beam. What color(s) can chlorophyll absorb?

The chloroplasts of the leaf have organized membranes (the thylakoids) which contain the molecules needed to convert light energy into chemical energy. When light shines on chloroplasts, ATP and NAPDH molecules will be formed.


Starch is an end product of photosynthesis produced during the light-independent reactions. Starch is easily detected: Lugol's Iodine Reagent, which is dark amber colored, turns blue black in the presence of starch (see Organic Molecules lab).

  1. Take the leaves that were boiled in alcohol, gently wash away the alcohol, and carefully flatten out each leaf in a Petri dish.

  2. Cover the leaf with Lugol's Iodine Reagent. After one minute gently wash away any excess iodine.

    • What color was the iodine solution before it was put on the leaf?

    • What color does the leaf become?
      A blue-black color indicates that starch has combined with the iodine in the test reagent.

    Compare the pattern of staining with the original pigment pattern sketched at the start.

    1. Does the leaf stain positively for starch all over? If not, what does this indicate about the photosynthetic activity in a Coleus leaf?

    2. Based on these tests, is the red pigment, observed originally in the Coleus leaf, a photosynthetic pigment?

    3. Is the green pigment a photosynthetic pigment?

    4. Look at the leaf under the microscope. Are there stained starch grains?

    Thanks to Profs. Lea Bleyman, Sy Schulman, and Emil Gernert for permission to adapt the Photosynthesis Exercise from BIO 1005 for use in BIO 1003.

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    Last updated 18 August 2019 (KD/JHW)