The Effects Of Colored Light On Photosynthesis

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Introduction

Photosynthesis is the process in which energy from sunlight is converted into chemical energy or simple carbohydrate. This chemical energy is the base for all food chains and its byproduct of oxygen provides a habitable environment for all living things on Earth. The process of photosynthesis occurs in two steps, the light reaction, and the Calvin cycle. The light reaction takes place in the thylakoid membrane of chloroplasts. Here, H2O is broken down into hydrogen and oxygen. During this step, NADP+, an electron acceptor, combines with hydrogen to reduce to NADPH (Johnson).

The Calvin cycle takes place in the stroma and does not require light energy, but instead uses ATP and NADPH created by the light reaction. The first step is carbon fixation, in which CO2 is attached to RuBP with the help of the catalyst Rubisco. This carbon molecule is then reduced to G3P, some of these G3P molecules are released to become glucose, while others are recycled to regenerated RuBP. For every 3 CO2 molecules that enter the Calvin cycle, only one G3P molecule is released. The entire process of photosynthesis can be summarized by the equation 6CO2 + 6H2O → C6H12O6 + 6O2 (Johnson).

Robert Hill provided groundbreaking research towards the understanding of photosynthesis in his 1937 study. The experiment itself simply showed that chloroplasts could perform the light reaction in water as long as there were light and an electron receptor present. The Hill reaction is what first led to the understanding that photosynthesis occurs in two steps, light-dependent and light-independent and that these steps can occur separately. It also showed that the oxidation of H2O, not CO2 as previously thought, is an integral part of photosynthesis. After H2O is oxidized, NADP+ bonds with H+ to reduce to NADPH. NADPH is then used during the Calvin Cycle for the synthesis reaction. In the experiment conducted by the lab group, DPIP was used in place of NADP+ because it functions as an electron acceptor and also changes from a blue color to a transparent one as it is reduced. This allowed us to measure the rate of photosynthesis by measuring the transmittance of light through a test tube containing DPIP, chloroplasts, buffer, and water. In the Hill reaction, the test tubes were only exposed to white light from a lamp with a flask filled with water to absorb heat. In the experiment performed, different colored light bulbs were used to test their effects on the rate of photosynthesis.

This variable was chosen due to the integral role of light energy in this step of photosynthesis. Different colors travel at different wavelengths and are therefore absorbed differently in chloroplasts. When measuring the transmission of light in the experiment, the spectrophotometer was calibrated to the wavelength of orange. This is due to the blue coloring of DPIP being the opposite of orange and therefore absorbing this color more efficiently. Following this logic, the opposite wavelength of green would fall between red and purple. If the chloroplasts are exposed to green light, then the rate of photosynthesis will be lower than that of other colored bulbs. The independent variables tested were green, yellow, blue, and white light bulbs with the dependent variable being the rate of photosynthesis.

Materials and Methods

Before starting the experiment, the chloroplasts had to be prepared by the TA. To begin, spinach was taken out of the fridge and destemmed. The spinach was kept cold to slow down photosynthesis and keep the leaves fresh, they were destemmed due to the lack of chloroplasts in the stem and the extra cellulose lowers transmittance. The spinach being left in the fridge led to degradation of the chloroplasts and so they were reactivated by being placed under light. Next, the TA removed a chilled blender and a chilled 0.5 M sucrose mixture. Once again, both were stored in the fridge to keep the chloroplast mixture as cold as possible to prevent photosynthesis from the beginning before the start of the experiment. The sucrose mixture was 0.5 M to be isotonic to the spinach and prevent the bursting or shrinking of chloroplasts that would be seen with a hypotonic or hypertonic mixture. Roughly 200 mL of the sucrose mixture was added to the blender along with the spinach and it was blended in three 10-second bursts. The spinach was blended to release the chloroplasts from the cell wall. The mixture was blended in short bursts to prevent the creation of heat that would speed up the photosynthesis reaction or denature the chloroplasts. The mixture was then strained through roughly 1 arms-length of cheesecloth to remove large macromolecules. It was then poured into light blocked bottles covered with electrical tape and stored on ice. This was to prevent a premature reservoir of high-energy electrons. During this preparation, the spectrophotometer was warmed up for 10 minutes at a wavelength of 605 nm. This wavelength, orange, was used because it is the opposite of blue on the color wheel and absorbs the most effectively.

Next, using the measurements in Figure 2, the test tubes were filled with the appropriate materials, saving the chloroplasts until just before measurement. The chloroplasts were only added just before to limit exposure to ambient light. The buffer maintained the proper pH level, the DPIP functioned as the electron acceptor, and the water was needed for the oxidation reaction. After the test tubes were assembled, the spectrophotometer was calibrated using the calibration tube. The calibration tube did not contain DPIP and so it calibrated the %Transmittance to exclude light blocked solely by the chloroplast mixture. Each tube was inverted to evenly mix and distribute the materials before each measurement. Next, each tube’s %Transmittance was recorded for the starting time. One of each tube was placed under the white, blue, yellow, and green lights respectively. An Erlenmeyer flask filled with water was placed in front of each light to absorb the heat created by the light bulb and prevent it from increasing the rate of photosynthesis. The calibration and dark tubes were also placed under the white light in a test tube rack. The other tubes were placed in a beaker to hold them up. To prevent ambient light exposure, the lamps were all placed under the desk and the tubes were transported to and from the spectrophotometer while covered in aluminum. The controls in this experiment were the tube kept under white light and the tube exposed to no light at all. The white light tube replicated the rate of photosynthesis under natural light and the dark tube controlled for no light exposure at all. This process of calibrating the spectrophotometer and measuring the %Transmittance of each tube was repeated every 3 minutes for 9 minutes total, inverting the tubes before each measurement. The rate of photosynthesis was then calculated using the formula (∆%Transmittance)/∆Time. This equation was used to standardize the measurements creating easier data to compare. The %Transmittance was used to calculate the rate of photosynthesis due to the blue coloration of DPIP. As DPIP was reduced to DPIPH, it became colorless and therefore transmittance increased. The reduction of DPIP was an indication that H2O was oxidized and the light-dependent reaction in the thylakoid membrane was completed. The rate of photosynthesis is important because plants are the primary producers and the base level of all food chains (Johnson). Photosynthesis must be performed at a rate fast enough to support the entire ecosystem. It is also important because it determines the level of oxygen in the air, improving breathing quality for humans and animals alike.

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Results

As seen in Figure 1, the %Transmittance increased for all tubes. This highest increase over time, as seen in Figure 4, was in both the yellow and blue tubes. Despite the dark tube starting at the highest %Transmittance, it had the lowest rate of photosynthesis (Figure 3). According to Figure 3, the yellow tube had the highest recorded rate of photosynthesis at 1.87 %T/Time, followed closely by the blue tube at 1.74 %T/Time. The white tube in this experiment served as a control to create a baseline for the effect of light color on the rate. As seen in Figure 5, the blue and yellow tubes greatly surpassed the white tube in the rate of photosynthesis while the green tube was slightly below.

Figure 4 also shows the mechanical error faced at the 3-minute mark. The yellow and green tubes inexplicably jumped to >100% transmittance but returned to normal at the 6-minute mark. This did not affect the tubes tested after the yellow and green. This error makes it difficult to observe the different slopes in Figure 4 but does not affect the overall rate of photosynthesis in Figure 5.

Discussion

The results of this experiment support the hypothesis. As seen in Figure 3, besides the control for no light, the green light tube had the lowest rate of photosynthesis at .32 %T/Time. This is most likely due to the chloroplasts' inability to absorb the green light and instead of reflecting the majority of it away. Recent studies have shown that green light could even inhibit photosynthesis from occurring rather than just happening at a slower rate (Folta and Munich). Blue and yellow are both adjacent to green on the color wheel allowing light to be absorbed more easily and leading to the highest rates of photosynthesis (Figure 3).

This variable of light color is mainly tested within the pigments in the spinach leaves. These pigments capture the light used for photosynthesis, the main one being chlorophyll a. Chlorophyll a is a blue-green color, is present in all photosynthetic organisms. Following the same logic as the hypothesis, the blue light tube could not have the highest rate of photosynthesis due to the blue pigment in chlorophyll keeping it from being fully absorbed. The different pigments in spinach also explain why the rate of photosynthesis increased for the green tube at all. There could be accessory pigments or carotenoids that present as a yellow or yellow-orange color making it more capable of absorbing green light and transferring the light energy to chlorophyll a where it is transformed into chemical energy for the light-dependent reaction.

Chlorophyll a then transfers this chemical energy to photosystem II for photophosphorylation. This energy causes an electron to jump to its excited state where it is then passed on to NADP+, or DPIP in this case, and replaced with an electron from H2O after it is oxidized. This high-energy electron that was excited by the chemical energy then travels down the electron transport chain and fuels chemiosmosis in photosystem I. The electron eventually reaches the reaction center where energy from more chlorophylls boosts it back up to its excited state. After traveling down another short electron transport chain and reduced NADP+ to NADPH, or in this case DPIP to DPIPH. The light-dependent reaction creates both the ATP and NADPH needed for the synthesis of G3 in the Calvin cycle.

The controls of this experiment represent both the normal photosynthesis reaction in plain white light and the photosynthesis reaction with only the light-independent reaction occurring. The white light tube is the closest to what is occurring in nature on an everyday basis, it provides a baseline for what is considered in this experiment the normal rate of photosynthesis. This allows us to assume that the rate of photosynthesis for the blue and yellow tubes is much higher than normal and that light is being absorbed at a much higher rate (Figure 5). The dark tube, on the other hand, demonstrates the need for light in the process of photosynthesis. After the tube was prepared, the chloroplasts were not further exposed to any more light. While the light-independent reaction can occur separately from the light-dependent reaction, NADPH and ATP are needed to complete the process. If the light reaction does not occur then no photosynthesis can occur either. This tube also controls for any event in which the color of DPIP would change for some other variable besides light.

One error in the data occurs at the 3-minute mark of both the green and yellow light tubes in Figure 1. For unknown reasons, the %Transmittance of these tubes jumped to above 100% (Figure 4). The spectrophotometer was calibrated using the calibration tube at the 3-minute mark and the tubes were tested before they yielded normal results. However, this data does not have a major effect on the final rate of photosynthesis as it is calculated using the change in %Transmittance over the full 9-minute interval, meaning only the first and last measurements were used. This would only affect the results if the rate of photosynthesis were to be calculated at the 3-minute interval also. This error was most likely the result of a mechanical malfunction and could have been avoided given the time to repeat the experiment.

Another error that could have affected the data is the placement of the test tubes in beakers to hold them up. Unfortunately, there were not enough test tube holders available to account for all test tubes and so the tubes were placed in glass beakers. This extra layer of glass could have prevented some amount of light from reaching the chloroplasts, lowering the rate of photosynthesis. Similar to this there could also be fingerprints on the test tubes themselves that would lower the %Transmittance measured by the spectrophotometer. The test tubes also could have been exposed to ambient light in the transition from under the desk to the spectrophotometer.

While natural sunlight will still be the primary light source for photosynthesis, this knowledge that different wavelengths may create a faster rate of photosynthesis could be applied to many agricultural practices. Many areas in the United States are home to food deserts, where fresh produce is not readily available. Being able to grow food faster and more efficiently can increase the availability of fresh produce and lower the price. This could consequently lower levels of obesity in food deserts (Ghosh-Dastidar, et al.). In areas of drought or other environmental factors that could lead to famine, crops could begin to be grown indoors. Using red or blue light would increase the rate at which crops grow, providing food in a much shorter amount of time. As our climate continues to change, growing environments and seasons will become less suitable for our current farming practices. Overall, the ability to adapt and provide an efficient way to produce food is essential to our future.   

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