laboratory

Assignment 6: Photosynthesis in Plant Cells

Downloads:
Work sheet for assignment 6
Work sheet as a pdf file (in case you cannot open a doc file)

Activities:
INTRODUCTION
ACTIVITY 1. REVIEW OF PHOTOSYNTHESIS AND CHLOROPLASTS
ACTIVITY 2. THE ROLE OF PIGMENTS AND LIGHT
Background
  Chromatography
Pigments and Photosynthetic Activity
ACTIVITY 3. MEASURING THE RATE OF PHOTOSYNTHESIS
  The spectrophotometer
  Performing the experiment
  Data collection
ACTIVITY 4. THE PLANT LEAF: AN ORGAN DESIGNED FOR PHOTOSYNTHESIS

Before attempting this exercise you should have a thorough understanding of the material in the topic Eukaryotes: Plant Cells.

Introduction

Most of the organisms familiar to us depend on the process of photosynthesis for two fundamental reasons: 1) photosynthesis fixes carbon to create larger organic molecules, and 2) this process releases oxygen into the environment. Thus, the world as we know it depends on the plants, protists and prokaryotes which are capable of photosynthetic activity. Photosynthesis is one of the few processes that can be used by organisms to tap inorganic sources of energy. Since all energy conversions that utilize organic sources (such as glucose) result in the some loss of energy (heat produced during metabolism), the living world would soon run out of energy were it not for the ability of photosynthesis to capture energy from the sun and store it in the organic molecules required by all living things. Additionally, organisms that utilize aerobic respiration to produce ATP require the oxygen that is produced by photosynthesis. In this laboratory, we will focus on both the photosynthetic process, including the unique pigments that absorb light energy, and the structures of plant cells and tissues that facilitate this energy conversion.

ACTIVITY 1. REVIEW OF PHOTOSYNTHESIS AND CHLOROPLASTS

To review the overall process of photosynthesis, use this relationships diagram. It is similar to the previous diagram that illustrated cellular respiration, except that instead of pull-down menus, you will pick the correct terms from a list and write them on your work sheet. When you have completed the table on your work sheet, answer question 1 in WebAssign. If this task proves difficult for you, go back and review the subtopic "What is Photosynthesis?".

As you know by now, photosynthesis occurs within the chloroplasts of eukaryotic cells. Chloroplasts are larger than mitochondria and can be seen more easily by light microscopy. Since they contain chlorophyll, which is green, chloroplasts can be seen without staining and are clearly visible within living plant cells. However, viewing the internal structure of a chloroplast requires the magnification of an electron microscope.

plant cells These living plant cells are viewed by light microscopy. Note the many green chloroplasts within each cell.
mitochondrion Here is a diagram showing the internal structure of a chloroplast. By now, you should be familiar with its parts and know the role
that each structure plays in photosynthesis. Note that the grana are stacks of individual units called thylakoids. Chlorophyll molecules are embedded within the thylakoid membranes.

Now, it is time to view a chloroplast by transmission electron microscopy. View this transmission electron micrograph of a plant cell, locate a chloroplast and capture the image for labeling. The micrograph is displayed as if using a "virtual electron microscope", so you will need to magnify the image and move to a region that contains the clearest view of chloroplast internal structures. Perform a screen capture of the chloroplast, then label a thylakoid, a granum, the stroma, and the outer chloroplast membrane. Submit your labeled image to WebAssign for question 2.

ACTIVITY 2. THE ROLE OF PIGMENTS AND LIGHT

Background

Wavelengths within the visible spectrum of light power photosynthesis. These wavelengths are only a part of a continuum within the electromagnetic spectrum; the shorter the wavelength the greater the energy.

spectrum
The Electromagnetic Spectrum

The light is absorbed by pigments contained within the chloroplasts of plant cells energizes electrons, raising them to a higher energy level. It is this energy that is used to produce ATP and to reduce NADP to NADPH. The release of energy from ATP and the oxidation of NADPH are then used to incorporate CO2 into organic molecules, a process called carbon fixation. Chlorophyll a is the most important photosynthetic pigment because it is directly involved in the conversion of light energy (photons) to chemical energy. For this reason chlorophyll a is called the primary photosynthetic pigment. It is present within the chloroplasts of all photosynthetic eukaryotes.

absorption All other photosynthetic pigments found in the chloroplasts of higher plants are called "accessory pigments". These include several other types of chlorophyll, the carotenoids and xanthophylls, and the phycobillins. The accessory pigments absorb light at wavelengths different from those absorbed by chlorophyll a and transfer part of that energy to chlorophyll a. Thus, the accessory pigments help to increase the efficiency of light utilization in photosynthesis.
Absorption spectra for selected pigments in Elodea (an aquatic plant)

When you understand the role of pigments and light in photosynthesis, answer questions
3 and 4
.

Chromatography

Because the different types of chlorophyll and other chloroplast pigments differ in molecular structure, they have different degrees of affinity for binding (absorbing) to the surface of fibers or particles. The practical application of differential binding properties of dissolved substances for purposes of their separation is called chromatography.

We will use paper chromatography to separate the pigments found in chloroplasts. Leaves of the Magnolia tree (found on campus) will serve as our model. We will separate the pigments found in the leaves of this plant according to their affinity for binding to cellulose fibers of paper.

First, a leaf is placed at one end of the paper. A blunt instrument (such as a coin edge) is drawn across the leaf, leaving a line of leaf matter on the paper. The paper is then placed in a covered jar with the application line at the bottom. A solvent (in this case ether:acetone) is added to the bottom of the jar so that it touches the bottom edge of the paper. The solvent slowly moves up the paper by capillary action. The leaf pigments will dissolve in the solvent as it crosses the application line and be carried up the paper with the solvent. The pigments are carried along at different rates because they are not equally soluble in the solvent and because they are differentially attracted to the fibers of the paper through the formation of intermolecular bonds, such as hydrogen bonds.
A developing chromatogram
 

Carefully observe the following video which illustrates the above procedure.

video - production of a chromatogram

The distance traveled by a particular compound (in this case leaf pigments) can be used to identify the compound. The ratio of the distance traveled by a compound to that of the solvent front is known as the Rf value; unknown compounds may be identified by comparing their Rf's to the Rf's of known standards.   

 rf  

Now view this photograph of the finished chromatogram. Use the photograph and the above information on Rf values to answer questions 5 and 6.

Not all the pigments that may be contained in plant cells are separated by this technique. Often refinements of this technique or the use of a different solvents or absorbent material may be needed to separate all pigments of interest. For example, in 2-dimensional paper chromatography,  a spot of material may be placed on the paper in one corner, leading to a narrow column of separation, then the paper is allowed to dry and placed at a 90 degree angle in a different solvent. The result is increased separation (better resolution) of pigments that are similar to each other in chemical structure. Using this procedure, more pigments can be separated than is achieved by 1-dimensional paper chromatography. Examine the example below, then answer question 7.

2-dimensional chromatograph
2-Dimensional paper chromatography

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