An Investigation of Light Dependent Reaction in Photosynthesis

Title

An investigation of Light Dependent Reaction (Electron Transport Chain) in Photosynthesis using dye DCPIP (2,6 dichlorophenol-indophenol)

Introduction

Photosynthesis is a very vital process that happens in the chloroplast which provides nourishment for all living organisms directly or indirectly. Photosynthesis is defined as the process of which the conversion of light energy from the sun to chemical energy that is stored in sugar and other organic molecules (Reece et al. 2011). There are two processes in photosynthesis that occurs in the chloroplast, namely the light dependent reaction and the light independent reaction.

The light dependent reactions occur in the grana of the chloroplast. Photolysis by which water molecule is split by the sunlight producing sources of electrons and H+ ions (protons) occurs here. The light dependent reaction also involves an Electron Transport Chain and two photosystems in the process of photophosphorylation which generates ATP. Photosynthesis starts with Photosystem II (P680) which contains photosynthetic pigments and chlorophyll that light energy. The light energy absorbed excites the electrons in P680 which are passed onto electron acceptors and electron carriers which forms the electron transport chain. The electrons lose energy and ATP is formed by chemiosmosis. Electrons are then excited again in Photosystem I (P700) and pass along electron transport chain ending with NADP+ as the final electron acceptor to form NADPH. Electrons from the photolysis of water will stabilize P680 and the cycle continues. ATP and NADPH formed are used to carry the energy and hydrogen into the light independent reactions that follows to produce sugar and other organic molecules(Taylor et al. 1997).

The blue dye, DCPIP is used in this experiment because it acts as an electron acceptor and can be substituted for plant's NADP in this system (Jones et al. 2007). The DCPIP will decolourise when reduced, hence indicating the rate or occurance of light dependent reaction in photosynthesis. The aim of this experiment is to investigate the various factors that affects the electron transport chain in light dependent reaction of photosynthesis. The factors involved investigated is the presence of light, boiled chloroplast, presence of enzyme inhibitor DCMU and nature of light(wavelength of green and red). The hypotheses for this investigation is that the DCPIP will only decolourise (be reduced) in the presence of light and light of wavelength region 650nm (Red) where to light reaction is complete. The rate of photosynthesis would also be highest in these condition. DCPIP will not decolourise in dark conditions and green light(wavelength region 510nm) and in the presence of enzyme inhibitor DCMU and boiled chloroplast. The electron transport chain will not function in the light reaction. The colour change of DCPIP will be determined by the absorbance value using a spectrophotometer.

Methods

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 contain 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 treatment immediately after addition of DCPIP and mixing 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 then placed horizontally on ice, under lights. At thirty minutes intervals, reading 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.

TREATMENT

BLANK

1 DARK

2 LIGHT

3 BOILED 4 DCMU

5 RED

6 GREEN

7

A chloroplast

suspension (ml)

1.5

1.5

1.5

-

1.5

1.5

1.5

B buffered sucrose

(ml)

5.5

5.3

5.3

5.3

5.2

5.3

5.3

C boiled chloroplast

suspension (ml)

-

-

-

1.5

-

-

-

D 0.01 M DCMU

(ml)

-

-

-

-

0.10

-

-

E DCPIP (ml)

(add this last)

-

0.20

0.20

0.20

0.20

0.20

0.20

Table 1. Experimental design for the electron transport experiment.

Results

ABSORBANCE

Time (mins) DARK Tube 2 LIGHT Tube 3 BOILED Tube 4 DCMU Tube 5 RED Tube 6 GREEN Tube 7

0 1.017 1.064 1.144 0.998 1.194 1.113

30 1.045 1.138 1.042 1.072 1.112