The purpose of this experiment was to observe whether the wavelength of light affected the rate of photosynthesis. In part one of the experiment my group set out to test which cell fraction contained the most chloroplasts. It was hypothesized that when we centrifuged the first pellet of cell fractionation it would be comprised of the larger organelles since they experience the largest centrifugal force and move the most rapidly; less time and force is required for them to separate. This led us to propose that pellet two would consist of the smaller organelles due to the fact that they require more time and force to separate from the supernatant. Since chloroplasts are smaller sized organelles, we also predicted that the higher concentration of them would be located in pellet 2 because it was centrifuged at an increased force and time. This hypothesis and prediction leads to the overall question; which cell fraction contains the most chloroplasts.
In order to obtain the cell fractionation with the most chloroplasts, my group prepared a cell homogenate from a piece of broccoli. We measure 40mL of that homogenate into the P1 tube and centrifuged at 600 x g for five minutes, then poured the supernatant from P1 into P2 and centrifuged P2 for an increased force and time. After centrifugation was completed we again poured the supernatant from P2 into the S2 tube to attain our three different cell fractions. To make the 12 cuvette concentrations we added certain amounts of isolation buffer to segregate the organelles in the solution, DCIP to indicate the rate of photosynthetic activity, and differing amounts of cell fraction to establish the variable in the experiment. We placed 3 cuvettes in the dark, one cuvette containing no cell fraction for a negative control and the remaining 9 cuvettes under normal light again with one cuvette containing no cell fraction for a positive control. In order to find which cuvette contained the greatest amount of chloroplasts we measured the absorbance of DCIP that was reduced in each cuvette at eight minute intervals using a spectronic 20 machine. We measured the absorbance of DCIP because it is indirectly correlated with photosynthetic activity due to its ability to capture the electrons that are transferred through the electron transport chain, causing the color intensity of its solution to decrease and reduce from blue to colorless. As the amount of DCIP decreases it signifies an increase in photosynthetic activity.
Graph 1
Graph one presents the overall change in absorbance of DCIP for each of the 12 cuvettes that were measured with the spectronic 20 machine. Results varied greatly with L4 reducing the most DCIP which we attributed to an experimental error, then L3 had the next greatest absorbance due to its concentration of the P2 cell fractionation. Our positive control reduced no DCIP since it did not contain any concentration of chloroplasts and our negative controls, placed in no light, reduced very little to no DCIP.
The results acquired through part 1 of this experiment provided support for our hypothesis that stated the second pellet would have a greater amount of chloroplasts, with the exception of the L4 cuvette. We speculated that this rapid reduction and inconsistency of results across our entire lab was due to another chemical that contaminated the cell fractionation and affected the amount of DCIP reduction. Even though all cuvettes showed photosynthetic activity, the L3 cuvette with P2 cell fractionation reduced the greatest amount of DCIP without experimental error. The L3cuvette’s consistent reduction of DCIP means that it contained the greatest concentration of chloroplasts entailing that the rate of water oxidation and DCIP reduction in photosystem II occurs at a faster rate compared to all other cuvettes tested. In order to insure that no other chemical components are present in the cell fractions, an additional experiment would be to centrifuge the cell homogenate for longer periods of time to ensure that a majority of the bigger contaminates are centrifuged out of the fractionation.
In part two of this experiment my group set out to test if the wavelength of light affected the activity of the chloroplast electron transport chains. We hypothesized that shorter wavelengths will result in greater amounts of excited electrons in photosystem II which in turn transfers this amplified amount of electrons to photosystem I for the process of reducing NADP+ to NADPH which is used in the formation of glucose molecules, overall increasing the rate of photosynthesis. Since blue light has the shortest wavelength we predicted that it would have the greatest photosynthetic activity due to its high energy.
Testing for which wavelength of light, red, green, or blue, reduced the greatest amount of DCIP was done by creating five cuvettes by adding the isolation buffer, DCIP and the P2 cell fractionation from part 1. We placed one cuvette in red light, green light, blue light, normal light for a positive control and no light for a negative control. In order to measure the photosynthetic activity we again measured the absorbance of DCIP of each cuvette using the spectronic 20 machine at six minute intervals.
Graph 2
Graph 2 has an overall decreasing pattern. The decrease correlates to the amount of DCIP reduction in each cuvette. Each cuvette began with a greater amount of DCIP than at the end of the experiment correlating with the increased amount of reduction of DCIP. Graph 2 shows that blue light had the most DCIP reduction, green light had the second greatest and red light reduced the least amount of DCIP.
Graph 3
Graph 3 displays the negative and positive controls of our experiment. The dark, no light, was our negative control and graph 3 shows an almost straight line for the absorbance of DCIP in no light. The normal light, positive control, decreased drastically and then tapered off to a constant line and absorbance of DCIP.
The results of part 2 of our experiment supported our hypothesis that stated shorter wavelengths will reduce the most DCIP and our prediction was also affirmed when the measurements recorded stated that blue had the most reduction of DCIP. One of the strongest reasons supporting that shorter wavelengths excite a greater amount of electrons than do longer wavelengths is the correlation between the absorption spectrum of certain pigments and the action spectrum of photosynthesis, the relative ability of wavelengths to support photosynthesis. These two spectrums produce a graph that shows the highest amount of light absorbance is between the 400-450 nm ranges which is the wavelength of blue light. One such pigment that absorbs light is chlorophyll. The energy absorbed from the light excites an electron from its ground level to an excited level. Through the passage of other chlorophyll molecules, this energy then reaches the reaction center of photosystem II. Photosystem II uses the light energy to oxidize two molecules of water into one molecule of molecular oxygen, one of the products of photosynthesis. The remaining electrons that were removed from the water will then travel via electron transport chain to photosystem I. In photosystem I these transferred electrons will replace the electrons that the light energy from the sun excites in the reaction center to ultimately reduce 2NADP+ to 2 NADPH (Miles). This molecule will carry the electrons to the carbon reactions of photosynthesis to produce glucose, the second product of photosynthesis. In conclusion, the shorter wavelength of blue light has a greater ability to excite more electrons sustain photosynthetic activity occurring at a fast rate.
An additional experiment I propose to carry out in order to see which pigment of light is absorbed the best by chlorophyll molecules is one that differs the intensities of each light. Now that my group concluded that blue light reduced the most DCIP out of red and green, I would want to know if each light was at the same intensity as each other. I would set up an experiment that differed the light intensities starting with green having the largest intensity to red having the next largest and then blue having the least and seeing if blue was still the light color that reduced the most DCIP or if the intensity of light plays a role in how much of the pigment is absorbed by the chloroplasts.
References
Asher, R. (2014). What Happens When a Chlorophyll Molecule Absorbs light? eHow. Retrieved from http://www.ehow.com/how-does_4922331_happens-chlorophyll-molecule-absorbs-light.html.
Leicht, B.G., McAllister, B.F. Project 1: Wet Lab 2B-Testing For the Activity of a Chlorplast Electron Transport Chain. 1411, Biology, University of Iowa. 2014.
Miles, B., Photosystems I and II. Texas A&M University. Retrieved from https://www.tamu.edu/faculty/bmiles/lectures/photosystems.pdf.