experiment to establish the link between light dependent and independent reactions

Practical Laboratory Experiment John Simmons

An experiment to demonstrate the link between light dependent and independent reactions in plant leaf matter.

Introduction

This practical experiment is to determine whether leaf material may be used to show photosynthesis.

In light dependent reactions, NADP is the reducing agent (it loses, or donates an electron and is oxidized) which is then used to produce reducing power for light independent reaction.

Isolating chloroplasts, the cell organelle which has the primary function is photosynthesis, will test this hypothesis, and subjecting it to lit and dark environments in order to record and analyse the results.

Solutions of other materials used in the experiment will also be tested as a control.

Method

The chloroplasts is isolated by grinding the leaf material (spinach),using a pestle and mortar, and the resulting pulp or brei, is sieved and equally divided into centrifuge tubes and separated by centrifugal force for ten minutes.

Next the supernatant, the residual liquid above the chloroplast pellet, is poured into a separate container and the chloroplast is suspended with isolation medium and remixed. This is then kept cold until use.

Five labelled tubes are then prepared using the suspended leaf material, numbered one to five.

DCPIP (2,6, Dichlorophenolindophenol), is a blue dye, acts as an electron acceptor and becomes colourless when reduced. Therefore, when this is added to isolated chloroplasts, any reducing agent produced by them may be detected.

The DCPIP should be used at room temperature.

Two of the tubes containing leaf material are placed under light and one in darkness. Readings are then recorded using a colorimeter, noting time and environment. Other tubes containing no chloroplast pellets are used as a control.

The table below (a) records the contents and environment of the tubes used in the experiment.

Table b below, records the colorimeter results of the five samples immediately after preparation.

b

c

Table c shows results after ten minutes in light environment.

Attached is a graph plotting these results by time versus absorption. The readings are numbered relating to the sample numbers on the tables above.

Analysis

Sample 1, which contained leaf extract and DCPIP and placed under a lamp, was seen to increase in absorption, indicating reduction of the DCPIP.

Sample 3, also containing leaf material but placed in darkness between recording, also increased in absorption.

Sample 4, the only other tube containing the isolated chloroplasts and also placed under lamp light, decreased in absorption. This sample, however, did not contain DCPIP, but was mixed with distilled water.

Sample 2 only contained isolation medium, and the reading on both occasions was the same.

Sample 5, containing supernatant and DCPIP, increased absorption, by a small amount.

From these findings, it has been shown that with the presence of chloroplasts and DCPIP, photosynthesis has taken place in a light dependent reaction. Also from the sample placed in the dark, light independent photosynthesis has also taken place.

If more time was allocated to the test, more accurate data would have been possible, and an extension to the experiment could have examined the effect over time on the sample placed in the dark, and how light deprivation would have affected the Calvin cycle.