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Comparing Photosynthesis in Red Cabbage, Green Cabbage, and Spinach Through DPIP Reduction
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Section 403

Jen Sallee

Andrew Clapper

February 25, 2007

Introduction

The scientist Robert Hill demonstrated in 1937 that chloroplasts in water were able to perform when light was present along with an electron receptor to release oxygen.  He gathered evidence that the oxygen given off had to come from water, because carbon dioxide was not present.  He also showed that there were both light dependant and light independent reactions.  Finally, his work indicated that oxidation-reduction reactions are a key part of photosynthesis. 

In plants and algae, photosynthesis occurs in an organelle called the chloroplast.  The chloroplast has two membranes that enclose the stroma, and a third membrane called the thylakoid membrane.  Within the thylakoid membrane are many photosystems surrounded by hundreds of light harvesting complexes, which are clusters of pigments and proteins.  During light-dependent reactions, photons, normally from sunlight, cause some photosystems to lose electrons.  The photosystem replaces the lost electrons with electrons from water molecules.  The water molecules separate into oxygen and hydrogen ions, and the oxygen leaves the cell.  The hydrogen ions build a concentration and electric gradient across the thylakoid membrane, and the gradients drive ATP formation.  In the light independent reaction, carbon dioxide diffuses into a photosynthetic cell, and the Calvin-Benson cycle turns six times in order to make a glucose molecule.  The light dependent and light independent reactions combine to give us the equation 12H2O (water) + 6CO2 (carbon dioxide) -----light energy and enzymes-- 6O2 (oxygen) + C6H12O6 (glucose) + 6H2O (water). 

The chemical compound dichlorophenol-indophenol (DPIP) can be used to determine the level of photosynthetic activity, because when it is near photosynthesizing chloroplasts, it accepts electrons and changes color from blue to colorless.  Therefore, we can measure the level of photosynthetic activity in a mixture of chloroplasts and DPIP by measuring the transmittance of light through the mixture.  This method allows us to explore the effect different conditions have on photosynthetic activity, including darkness and boiling of the chloroplasts. 

We observed with a compound microscope that the chloroplasts in an elodea cell seem to provide the plant leaf with its green color.  We also observed that leaves of iceberg lettuce are less of a dark green color than spinach leaves.  Likewise, the leaves of a red cabbage plant appear to have no green pigment.  In order to measure photosynthetic activity in a chloroplast-DPIP mixture, a spectrophotometer can be utilized.  A spectrophotometer measures the amount of light transmitted as it passes through a solution.  If more photosynthetic activity is occurring in a solution, the spectrophotometer will indicate a higher amount of transmitted light, because the DPIP will have been reduced so that it allows more light to pass through the solution.  During a previous study, this method was used to observe the photosynthetic rate of spinach chloroplasts.  Comparison with a chloroplast solution that was kept in the dark showed that the majority of photosynthetic activity in spinach chloroplasts is light dependant.  Also, a sample of chloroplasts was boiled and compared to a sample that was unboiled in order to show how heat can denature the proteins in chloroplasts that are essential for the light dependent reactions. 

We hypothesize that the chloroplast from the iceberg lettuce will show less transmittance than the spinach chloroplast because chloroplasts are less abundant and less active in the iceberg lettuce, and therefore will cause the DPIP to reduce less.  Also, we hypothesize that the chloroplast from the red cabbage will show less transmittance than the iceberg lettuce, for the same reasons.   

 

Method

A specific set of actions were used to prepare the chloroplasts for the samples to be used in the experiment.  Spinach leaves were placed under a lamp in order to activate the chloroplasts in the leaves while the osmolarity fluid was prepared.  Point five sucrose osmolarity fluid was used for the experiment to provide an environment for the chloroplasts that was neither too hypertonic nor too hypotonic.  The osmolarity fluid was poured up to the top of the blades of the blender.  The stems were removed from the spinach leaves because they contain few chloroplasts.  The top of the blender body was placed on the body and the blender was activated in ten second bursts to keep the contents from heating, because heating would de-nature the proteins in the chloroplasts and cause them to no longer function normally.  The purpose of blending the spinach leaves was to free the chloroplasts from the cells they were contained in.  Two layers of cheese cloth were placed on top of a beaker.  The contents of the blender were then poured through the cloth in order to filter out large pieces of leaf matter that might interfere with the spectrophotometer readings.  The liquid was then poured into a test tube and transferred to small plastic containers for each group of experimenters, and placed in a container with ice to keep the chloroplasts cool.  Again, the chloroplast samples were kept cool in order to protect the structure and functionality of the proteins in the chloroplasts, so that the chloroplasts would function normally.  The spectrophotometer was set to six hundred five nanometers wavelength light, which is an optimal wavelength to measure DPIP reduction with.

            The experiment components were one test tube rack, one goose neck light, four spectrophotometer tubes (cuvettes), an ice container with ice, four one ml transfer pipettes, at least sixteen Kimwipes, at least 52 ml distilled water, at least 16 ml .1 -M phosphate buffer, 64 pieces of parafilm, and one large Erlenmeyer flask filled with water to act as a heat sink.  The test tube rack was used to hold the cuvettes when they were not being measured.  The goose neck light provided light to activate photosynthesis in the chloroplast samples.  The cuvettes contained the solution that was used to measure the DPIP reduction caused by photosynthesis.  The ice container and ice kept the chloroplast samples cool.  The pipettes were needed to measure precise amounts of liquid to be placed into the cuvettes.  The parafilm pieces kept the contents of the cuvettes contained while the cuvettes were inverted during mixing. 

            We numbered the cuvettes one through four.  Cuvette one was used as the calibration tube, cuvette two was used for the spinach chloroplasts, cuvette three was used for the red cabbage chloroplasts, and cuvette four was used for the iceberg lettuce chloroplasts.  The cuvettes were handled only by the top portion in order to keep the sides free from finger prints.  They were also handled with care because they were specially made for spectrophotometers.  The Kimwipes were used to clean the outside of each cuvette before each measurement with the spectrophotometer.

            A container of spinach chloroplasts, a container of red cabbage chloroplasts, and a container of iceberg lettuce chloroplasts were obtained from the instructor and put on ice.  One ml of buffer was added to each cuvette with the buffer pipette.  Four ml of distilled water was added to the calibration tube with the water pipette, and three ml of distilled water was added to each of the other tubes with the water pipette.  One ml of DPIP was added to cuvettes two, three, and four with the DPIP pipette. 

            Three drops of chloroplasts were added to the appropriate tubes with the corresponding pipette.  Parafilm was immediately placed on the tubes, and the tubes were turned upside down once to mix the contents.  The tubes were then cleaned with a Kimwipe.  Next, the calibration tube was quickly placed into the sample holder of the spectrophotometer.  The tube was aligned with the appropriate mark.  The lid of the sample holder was closed, and the spectrophotometer was adjusted so that it read one hundred percent transmittance.  This was done by turning the 100%T/ zero A dial (the lower left dial) until it read 100%.  Next, the cuvette was removed, the sample holder lid was closed, and the next three cuvettes were read. 

Cuvette was read by quickly placing it into the sample holder, closing the sample holder lid, and immediately reading the transmittance.  No dials were turned during this step.  Cuvettes three and four were read with the same process.  The contents were mixed and the outside of each tube was dried before each reading.

The readings of the spectrophotometer were recorded as results in a data chart.  The first set of readings was labeled as time zero.  Each cuvette was mixed and read again at five, ten, and fifteen minutes.  The calibration tube was read each time first.  Before each interval, the calibration tube was used to calibrate the spectrophotometer to 100%.  In between readings, the tubes were placed in the test tube rack.  The flask with water was always in between the test tube rack and the goose neck lamp.  Light from the lamp reached the test tubes, while the flask with water acted as a heat sink.  A data chart was drawn in the space provided in the lab manual, and the data of the team was recorded there.  One axis of the chart was used to designate the measurement times of zero, five minutes, ten minutes, and fifteen minutes.  The other axis was divided between the calibration tube, spinach, red cabbage, and iceberg lettuce.  In each resulting square, the transmittance percentage displayed by the spectrophotometer was recorded.   

 

Results

The DPIP mixed with the spinach chloroplasts was reduced more than both the DPIP mixed with the chloroplasts from the red cabbage and the DPIP mixed with the chloroplasts form the iceberg lettuce, which caused the transmittance rate to be highest in the solution from the spinach chloroplasts (Table 1).  The chloroplasts from the spinach were more active than either the red cabbage or iceberg lettuce chloroplasts (Table 2).  The red cabbage solution showed a greater overall rate of photosynthesis than the iceberg lettuce solution (Table 2).  The rate of photosynthesis of the spinach solution was many times higher than the rate of photosynthesis for either the red cabbage solution or the iceberg lettuce solution (Table 2).  The periodic and overall rate of photosynthesis for each solution is displayed in Table 2.   

 

Discussion

Based on the data gathered and displayed in Table 2, we have evidence that supports the hypothesis that the spinach chloroplasts will have the greatest rate of photosynthesis, because the DPIP that was mixed with the spinach chloroplasts was reduced the most (Table 1).  However, according to the data in Table 2, the hypothesis that iceberg lettuce chloroplasts will have a greater rate of photosynthesis than red cabbage chloroplasts should be rejected, because the red cabbage chloroplasts appear to have had a higher rate of photosynthesis. 

Several factors may have affected the strength of the findings.  The weight to volume ratio in each plant may be different.  If this is true, a different amount of chlorophyll solution is required for each cuvette in order to have the same number of chloroplasts.  It is possible that red cabbage and iceberg lettuce need to be exposed to light for a longer period of time than spinach in order for the chloroplasts to become active.  Human error may have resulted in an incorrect measurement of DPIP, water, buffer, or drops of chloroplast in the cuvettes.  It is possible that red cabbage and iceberg lettuce have longer life spans or slower growth rates than spinach, both of which might result in lower rates of photosynthesis.  The control used in our study, the photosynthetic rate of spinach, provided us with evidence that the method of using DPIP reduction to assess photosynthetic activity is sound.  It also gave us evidence to allow us to compare the photosynthesis rate of spinach chloroplasts to the photosynthesis rates of red cabbage and iceberg lettuce. 

Appendix:Table 1
Transmittance
Minutes Calibration  Spinach Red Cabbage Iceberg Lettuce
0 100.00 31.00 25.50 22.50
5 100.00 45.80 25.70 22.20
10 100.00 59.30 25.60 22.30
15 100.00 71.60 25.70 22.40
*Readings are percent transmittance with 605 nm wavelength light.
Appendix: Table 2
Rate of Photosynthesis
Minutes Calibration Spinach Red Cabbage Iceberg Lettuce
0 0.00 0.00 0.00 0.00
5 0.00 2.96 0.04 -0.06
10 0.00 1.35 -0.01 0.01
15 0.00 0.82 0.01 0.01
Overall 0.00 2.71 0.01 -0.01

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