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INVESTIGATING LIGHT-DEPENDENT ELECTRON TRANSPORT USING DCPIP


Photosynthesis plays an integral role in providing life for many ecosystems. It is well
known that photosynthesis is the process where plants assimilate CO2 to form sugars and more
complex compounds that are required for growth. Photosynthesis consists of two parts: the
light-dependent process and the light-independent process. The light-dependent process of
photosynthesis turns the energy from the sunlight into chemical energy. This process occurs in a
multiprotein complex called photosystem II (PSII) and photosystem I (PSI) which are found
embedded in the thylakoid membrane. The light-independent process is also known as the
Calvin Cycle where the NADPH and ATP produced in the light-depended process is used to
make carbohydrates.
The focus of this experiment is the electron transport chain within the light-dependent
process and how it uses redox reactions to generate ATP. The aim is to determine what factors
affect the rate at which photosynthesis occurs by measuring the reduction of DCPIP.
It is hypothesised that the chloroplast solution left in the dark will have no change in
absorbance since light is required for the reaction to take place. Since there are enzymes within
the chloroplasts, the boiled suspension will have no in absorbance due to the denaturation of
these enzymes. When DCMU is added, there will also be a small decline in absorbance. If the
suspension is exposed to only red light there will be a large decline in absorbance whereas
when the suspensions are exposed to only green light, there will be a small decline in
absorbance.
Method
Seven spectrophotometer tubes were numbered and solutions A-D were added
according to the volumes shown in Table 1. Tube 1 was capped and inverted several times. The
spectrophotometer was calibrated using Tube 1, which contained chloroplasts and sucrose only,
as the blank, to ensure that any changes in absorbance for the other treatments could be
attributed to the reduction of the dye DCPIP. At time zero (mins), absorbance was recorded for
all treatments immediately after addition of DCPIP and mixing of contents. Immediately
following the time zero reading, tube 2 was wrapped in foil and tubes 6 and 7 were placed into
larger tubes covered in red and green cellophane respectively. Tubes 1-5 were also placed into
larger tubes. All tubes were then placed horizontally on ice, under lights. At fifteen minute
intervals, readings of absorbance were taken for all treatments, except for the dark tube which
was kept wrapped in foil for 60 minutes, after which its absorbance was measured.

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Table 1: Experimental design for the electron transport experiment.

BLANK
1

DARK
2

TREATMENT
LIGHT
BOILED
3
4

DCMU
5

RED
6

GREEN
7

chloroplast
suspension (ml)

1.5

1.5

1.5

1.5

1.5

1.5

buffered sucrose
(ml)

5.5

5.3

5.3

5.3

5.2

5.3

5.3

boiled chloroplast
suspension (ml)

1.5

0.01 M DCMU
(ml)

0.10

0.20

0.20

0.20

0.20

0.20

0.20

DCPIP (ml)
E

Results
After compiling all the results into one graph, it can be seen that there are many factors
that can affect the rate that photosynthesis occurs. There were 5 different treatments that were
applied to the chloroplast suspensions and it was found that the dark, boiled and DCMU
treatments severely hindered the rate of photosynthesis. On the other hand, the red and green
light treatments still allowed photosynthesis to occur although at a slower rate than the
chloroplasts that were only exposed to white light (Figure 1).

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0.9
0.8
0.7
0.6
0.5

Absorbance

0.4
0.3
0.2
0.1
0
0

10

20

30

40

Time (minutes)

50

60

70

Linear ()
Linear ()
Dark
Light
Light
Boiled
Linear (Boiled)
Boiled
DCMU
Linear (DCMU)
DCMU
Red light
Red light
Green light
Green light

Fi
gure 1: The graph showing the absorbance of a solution over time for different chloroplast
treatments.

Discussion
The purpose of this experiment was to determine what factors affect the rate at which
photosynthesis occurs by measuring the reduction of DCPIP. It was predicted that the
chloroplast solution in Tube 1 that was left in the dark will have no change in absorbance. The
results do support the hypothesis since light is required for the reaction to take place and
therefore without light, the reduction of DCPIP is not able to take place. Similarly, in Tube 4, it
was predicted that the boiled chloroplasts will result in no change in absorbance because of the
heat denaturing the enzymes within the membranes of the chloroplasts. This was not the case
since there was a slight decrease in absorbance which was likely due to the fact that not all
enzymes were denatured by the heat which allowed some electron transfer to occur. Tube 5 had
similar results to Tube 4 which supports the hypothesis that there will be a small decline in
absorbance. The herbicide DCMU was added to this chloroplast suspension which inhibits the
electron transport chain between PSII and PSI, preventing DCPIP from being reduced.
On the other hand, Tubes 6 and 7 showed more dramatic decreases in absorbance.
Tube 6 was exposed only to red light and it was hypothesised that there will be a large decline
in absorbance which held true due to the fact that chloroplasts will readily absorb red light
however due to the limited absorption spectrum, the absorbance did not decrease as rapidly as
the chloroplasts exposed to white light. Tube 7 was exposed only to green light and it was
predicted that there will also be a slow decline in absorbance. The results do support this
hypothesis due to the fact that chloroplasts reflect green light therefore when only green light is
shone on the chloroplasts, it dramatically limits the absorbance spectrum but also still allows
some light to be absorbed.
A source of error for this experiment was that some of the test tubes were wiped down
with tissue paper instead of kimwipes before they were placed into the spectrophotometer. This

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would have caused some absorbance readings to be higher than expected due to the particles
left on the test tubes. Another source of error was that Tube 6 (red light treatment) was initially
left in the light for too long before it was placed in the Falcon tube, therefore its initial
absorbance was lower than the other test tubes.