laboratory
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

Pigments and photosynthetic activity

The rate of photosynthesis is often measured at different wavelengths of light, using the same sample of plant pigments. The graphic representation of this is known as an action spectrum. The action spectrum for photosynthesis by a particular plant can be compared to the absorption spectrum for the same plant give us a clue as to the role of accessory pigments and whether these pigments are important in photosynthesis.

When light strikes an object several things can happen.  The light can be absorbed, reflected or transmitted. 

eyes and light

The colors that we see are those wavelengths which were not absorbed, but were transmitted or reflected back to our eyes and interpreted by our brain as color. The graphic representation of the relative pattern of absorption is known as an absorption spectrum. We have already seen the absorption spectra of chlorophyll a, b and carotene in Elodea, a common water plant. The absorption spectra for chlorophyll will not vary much from plant to plant. The chlorophylls always absorb more of red and blue wavelengths and much less of the green, thus appearing green to our eyes. But the action spectra of photosynthesis will vary from plant to plant because of the contribution of various accessory pigments to photosynthesis.

The the action spectrum below was obtained from Elodea. Compare the action spectrum to Elodea's absorption spectrum, then answer question 8.

action spectra An action spectrum for photosynthesis plots the rate of photosynthesis against the wavelength of light.
Action spectrum for Elodea leaves
 

ACTIVITY 3. MEASURING THE RATE OF PHOTOSYNTHESIS

As the pigments that we just examined absorb light, electrons within photosystems are excited to a higher energy level. These high-energy electrons are essential to ATP production and reduction of NADP+ to NADPH.  Both ATP and NADPH are necessary for carbon fixation.

DPIP
In the following activity, chloroplasts are isolated and their membranes disrupted. In place of NADP+, which is no longer readily available to the electron transport chain,
2.6-dichlorophenol-indophenol (DPIP) is added to the solution and acts as a final acceptor for the energized electrons. When DPIP is reduced, it changes from blue to colorless. Thus, by using DPIP in an assay, we can subject the isolated thylakoids to different conditions and use the color changes we observe as an indicator of relative photosynthetic activity .   
Changes in the color of DPIP as it is reduced (accepts electrons)  

The spectrophotometer

As the DPIP is reduced and becomes colorless, the resultant decrease in light absorption can be measured over a period of time using a spectrophotometer. We will use the same spectrophotometer as for the previous experiment on mouse respiration. All spectrophotometers generate a source of light which is broken up into various wavelengths, some of which are allowed to pass through a sample solution where some of the light may be absorbed. After passing through the sample, the remaining light is detected by a sensor. The electronics of the spectrophotometer enhance and analyze the light signal in various ways before displaying a measurement. More complex spectrophotometers can also produce a graph of measurements over time.

spectrophotometer components
A light source is split (in the example by a prism).  Selected wavelengths are allowed to pass through a sample and the light is measured by a sensor and interpreted to give a reading of relative light absorption.

Setting up the spectrophotometer:
Since DPIP is a deep blue color, it should absorb red wavelengths (those wavelengths at the opposite end of the spectrum).  Thus, we have chosen to measure absorption at a wavelength of 610 nm.

1. You will first set the machine for 0 absorption, using water (a colorless sample).

2. Then you will measure the absorption of a solution containing DPIP and all components found in the experimental mixture except for the chloroplast thylakoids. Since we are measuring absorption of a deep blue substance that will lose color during the assay, absorption will decrease with time. Color change will begin immediately when chloroplasts are added to the DPIP solution. Thus, we will use the tube containing all assay components except thylakoids to set the value of the zero time-point (absorption of the DPIP solution before photosynthesis begins).

View the following video to see our technician performing the above steps.

video - setting up the spectrophotometer

Before beginning the experiment, a solution of thylakoids must be obtained. Our technician will prepare this solution from spinach leaves. Watch the following video to see how this is done.

video - preparing a solution of thylakoids

Before proceeding to the photosynthesis experiment, answer questions 9 and 10.