Investigate Light-Dependent Electron Transport Using Dcpip

INVESTIGATE LIGHT-DEPENDENT ELECTRON TRANSPORT USING DCPIP

Introduction:

Photosynthesis is a vital process that occurs in plants, as it is the system that enables its autotrophy. In photosynthesis, light energy is absorbed the by pigments in light-harvesting chemicals, which is chlorophyll, found in the organelle chloroplasts. This energy is transferred into subsets in photosystem I (PSI) and photosystem II (PSII), where splitting of water molecules and consequent transfer of electrons through redox cofactors ultimately reduces NADP+  to NADPH. (Davis et al. 2015).  The passing of electrons along the electron transport chain is a spontaneous reaction, and thus relies on the energy level of the electrons to move down the chain. For the electrons to be ‘excited’ or energised, light is required. The aim of this experiment was to investigate how the rate of photosynthesis is affected by varying the conditions exposed to the to the chloroplasts of silverbeet leaves. For the convenience of the experiment, DCPIP was used as a synthetic electron acceptor. The coloured dye becomes colourless when it accepts the electrons produced during the light reactions. Thus it was hypothesised that the test tube that was blocked from light completely would have the highest absorbance readings, (and hence the slowest rate of photosynthesis), followed by the ones containing DCMU, boiled chloroplast and covered in green cellophane. The test tubes exposed to white light and covered in red cellophane were hypothesised to have decreased in absorbance reading the most and therefore having a faster rate of photosynthesis.

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 fading 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.”

Results:

The initial and final absorbance readings from the spectrophotometer showed that over the 60-minute period, there was an absorbance decreace of 0.011 for the test tube that was not exposed to any light. For the test tube exposed to normal light, there was a decrease of 0.685. For the test tube containing boiled chloroplast, the absorbance increased by 0.044. For the test tube containing the electron transport inhibitor DCMU, the absorbance also increased, by 0.023. For the test tube covered in red cellophane, the absorbance dropped by 0.551. Lastly, for the test tube covered in green cellophane, the absorbance dropped by 0.362. 

Figure 1. Change in absorbance of silverbeet chloroplasts treated with DCPIP dye over a 60-minute time period, for various conditions

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Discussion:

The hypothesis that the photosynthesis rates would be in increasing order of the test tubes exposed to no light, containing DCMU, boiled chloroplast, covered in green cellophane, covered in red cellophane and exposed to white normal light was supported, except for the conditions of the test tube containing DCMU and the test tube containing the boiled chloroplast. The absorbance readings for the conditions with DCMU, overall increased, perhaps due to experimental errors such not inverting the test tube enough, so the solution didn’t mix properly. What was predicted for this condition was little to no change in absorbance over the 60-minute period, as the DCMU would have inhibited the electron transport process, which is imperative for the light reaction to occur, and for photosynthesis to proceed. Throughout the 60-minute period, overall, the test tube that was exposed to no light had a drop, albeit slight, in absorbance. It was predicted that there would be little to no change in absorbance due to this treatment, as light is required for photosynthesis to occur. Perhaps the slight drop in absorbance, which is indicative of photosynthesis, is due to the exposure to the beam of white light while the test tube was in in the spectrophotometer. The test tube that was covered in green cellophane had a smaller drop in absorbance overall, when compared to the test tube covered in red cellophane. This was a predicted result, as chlorophyll absorbs red and blue-violet light. Light absorption by this wavelength can enhance the yield of photosynthesis (Rabinowitch 1960). On the other hand, “Chlorophyll has minimal absorption of green light” (Snowden 2016), which means that there would not be enough energy to excite and energise the electrons to go through the electron transport chain, and therefore no initiation of the process of photosynthesis.  The test tube that demonstrated the fastest rate of photosynthesis was the one with the condition of being exposed to normal white light. This was also an expected result, as white light includes both violet-blue and red light, and thus it would be absorbing more energy than the test tube, which was restricted to only red light by being covered in red cellophane. By absorbing more light, the chloroplast would have more excited electrons which were able to pass through the electron transport chain. This would make the photosynthesis process relatively faster than all the other photosynthesis processes occurring in the test tubes with differing treatments.