Aim:

To investigate the effect of different temperatures on the rate of light-dependent reaction of

photosynthesis in spinach leaves using the dye DCPIP.

Introduction:
In the light-dependent reaction of photosynthesis, NADP+ acts as the final electron acceptor of
cyclic photophosphorylation.
DCPIP can also accept electrons from ETC and be reduced, turning from blue to colourless.
Hence, DCPIP competes with NADP+ for electrons.
Rate of photosynthesis (of the light-dependent reaction) is determined by measuring the amount
of DCPIP present in the spinach chloroplast cells after 1 minute, using a spectrophotometer.

Variables:
1) Independent variable: temperature of the solution at 5 different temperatures (5C, 15C,
25C, 35C, 45C)
2) Dependent variable: absorbance of the chloroplast solutions after 1 minute
3) Controlled variables:
Carbon dioxide concentration (atmospheric CO2)
pH of solution (buffer is added to ensure constant pH throughout the experiment)
volume of DCPIP added (5 drops)
volume of chloroplast solutions in each test tube (1cm3)
Time of exposure (1 minute)
light intensity (distance between the lamp & capillary tube at 5cm)

Procedure:
1. Cut 5 fresh spinach green leaves with a sharp knife and homogenize with cold buffer solution
with mortar and pestle and fine sand.
2. Cover the chloroplast extract with a light-proof aluminium foil and placed in a petri dish.
Extract has to be maintained at low temperature using ice.
3. Using pipette, add 5 drops of DCPIP to the leaf extract in the petri dish. Rock the petri dish to
mix the liquids and replace the foil cover
4.Take 5 test tubes.
5. Pipette out about 1 cm3 of leaf extract/DCPIP mixture into each test tube and cover the test
tubes quickly in aluminium foil to prevent exposure to light.
6. Cool the first test tube by placing them in a water bath containing ice, and maintain the
temperature at 5C for 1 minute.
7. Place the test tube 5 cm away from the lamp (measured by a ruler), remove the aluminium
foil from the tube and immediately turn on the lamp.
8. After one minute of exposure to the lamp, measure and record the absorbances of the
chloroplast solutions in the test tube using a spectrophotometer at a wavelength of 600 nm.
9. Steps 6-8 are repeated for the subsequent 4 test tubes with a water bath ensuring
temperatures of 15C, 25C, 35C and 45C respectively.
10. Set up in replicates of 3 for each temperature.

Diagram:

Results:

Test Temperature/ Absorbance after 1 minute/ % Rate of

tubes C Photosynthesis/
s-1
1st trial 2nd trial 3rd trial Average

1 5

2 15

3 25

4 35

5 45

Graph:
Evaluation:

A curve graph that should show an increasing gradient from 5C to 25C, peaking at 35C,
before steeply decreasing after 35C.

This is as temperature increases,the rate of light dependent reaction of photosynthesis

increases, with the optimal temperature at 35C. After 35C, enzymes involved in the
light-dependent reactions are denatured by the relatively high temperature, and hence, this
decreases the rate of photosynthesis.