Photosynthesis in plants is affected by the intensity of the light the plant is exposed to. For this experiment, DCPIP was added to cuvettes with spinach chloroplasts, which were exposed to an incandescent light at different distances for different intervals of time. After each exposure, the cuvettes were placed in a spectrophotometer set at 621 nm and the absorbance of DCPIP was measured. At 37 mm away from the light, the absorbance decreased at 0.033 absorbances/minute. At 27 mm away from the light, the absorbance decreased at 0.043 absorbances/minute. At 5 mm away from the light, the absorbance decreased at 0.050 absorbances/minute. As the distance from the light increased, the absorbance readings on the spectrophotometer went down at a faster rate. This means that the amount of DCPIP in the chloroplasts decreased quicker as the intensity of light was higher, and that photosynthesis occurs more quickly or slowly depending on the intensity of the light.
Photosynthesis is the process that allows plants to survive (Chiras, 1993). It provides ATP (which can be used for energy), starch, cellulose, fats, and nucleic acids among other large molecules, and it consumes CO2 to produce O2 (Chiras, 1993). The plant chloroplast cell consists of an inner and outer membrane (Thorpe, 1984). Inside of the cell consists of chlorophyll and stroma. The stroma is a fluid substance comparable to cytosol in animal cells. Chlorophyll are particles that absorb light and pass the energy obtained onto thylakoid disks (Thorpe, 1984). Situated in the membrane of the thylakoid membrane is the electron transport chain. Along the electron transport chain, two electrons from H2O are excited by light in Photosystems I and II to reach a higher energy level. This energy is used to convert NADP+ to NADPH and to drive electrons into to thylakoid space, which creates a gradient. This gradient fuels ATP Synthase, which converts ADP + Pi to ATP. ATP is used for energy in many processes in plant cells. In the experiments performed, DCPIP was added to chloroplast cells, which replaced the NADP+ along the electron transport chain. Using a spectrophotometer, the amount of DCPIP present was able to be determined and tests using variable intensities of light were performed on spinach chloroplast cells.
Materials and Methods
First, a room was insulated from light and the lights were turned off. A green light was used in order to see. Six cuvettes were then obtained and labeled with the numbers 1 through 6. Tube 1 consisted of 0.5 mL chloroplast, 3.0 mL cold buffer, 1.5 mL cold distilled water, and 0.0 mL of DCPIP, which were all dispensed into the tube using a micropipettor. This tube was used as a blank for the spectrophotometer which was set to 621 nm. Tube 2 was used as a control and was covered completely with tin foil in order to insulate it from light. Tube 2 and 3 were filled with 0.5 mL chloroplast, 3.0 mL cold buffer, 0.5 mL cold distilled water, and last with 1.0 mL DCPIP. Immediately after being filled with the DCPIP and agitated, the tubes were both placed in the spectrophotometer and their absorbances were recorded. Tube 2 was first removed from its foil before being put in the spectrophotometer, and it was put back on after the reading. They were than placed 11.5 mm away from an incandescent light for 3 minutes. Their absorbances were again recorded and this was repeated until the control tube came to a constant reading while tube 3 gradually went down. Tubes 4 through 6 were also filled with 0.5 mL chloroplast, 3.0 mL cold buffer, 0.5 mL cold distilled water, and 1.0 mL DCPIP right before being subject to light. Tube tubes were placed 37 mm, 27 mm, and 5 mm away, respectively. Their initial absorbances were recorded and following absorbances were recorded every 60 seconds for 6 minutes was exposed to the light.
|Reading||Distance (in mm)||Time (in minutes)||Absorbance|
|Tube 2 (Control)||11.5||0||0.44|
|Tube 2 (Control)||11.5||3||0.44|
|Tube 2 (Control)||11.5||6||0.44|
|Tube 2 (Control)||11.5||9||0.44|
|Reading||Distance (mm)||Rate of Decrease (Absorbance/min)|
Tubes 2 and 3 were used to prove that it was indeed the light causing the absorbance to go down, and not the heat from the light. Tube 2 was covered with foil to prevent it from being exposed to light and its absorbance stayed constant, while the absorbance from tube 3 which was uncovered went down. As the tubes were placed closer to the light, their absorbances went down quicker, which was expected. The absorbance of tube 6 went all the way down to 0.16, which seemed very low (Table I). Test tube 5’s absorbance went low also, going down to 0.21 (Table I). Test tube 4 had very sporadic readings (Table I). This may be attributed to it not being thoroughly mixed enough.
The absorbances readings recorded measured the amount of DCPIPoxidized in the cells. As photosynthesis occurred, electrons were donated to the DCPIPoxidized, forming DCPIPH2 reduced. DCPIPoxidized is able to absorb light from the spectrophotometer at 621 nm, hence that it why it was calibrated at 621 nm. As photosynthesis took place, there was less and less DCPIPoxidized available for absorbance. That is why the absorbance readings went down over time.
Bidwell (1979) reported that light absorption is not really affected by temperature. The results gained from the experiment were consistent with his findings. When tube 2, which was covered in tin foil, was exposed to the light, its absorbance stayed constant. Though light was not affecting the tube, it could still be heated up. Because its absorbance did not move, this showed that the heat did not affect any of the absorbance readings for any of the test tubes.
Tubes 3 through 6 were all exposed to the light and their absorbances went down over time. The rate at which their absorbances went down increased as their distance from the light source decreased. The intensity of light directly affects the rate of photosynthesis (Bidwell, 1979). Graphs show that the higher the intensity, the higher rate of photosynthesis. The intensity in this experiment was increased by moving the tube closer to the light.
Lastly, the reason a green light was used in order to see was because chlorophyll absorbs all colors of light except for green and yellow (Chiras, 1993). Additional experiments using different colors of light or different light bulbs would be interesting, as the chlorophyll trap different kinds of lights at different rates. This could show what range of the color spectrum chlorophyll accept best.
Bidwell, R. G. S. 1979. Plant Physiology. (MacMillian Publishing Co., NY, NY) 726 p.
Chiras, Daniel D. 1993. Biology: The Web of Life. (West Publishing Co., St. Paul, Mn) 896 p.
Thorpe, N. O. 1984. Cell Biology. (John Wiley & Sons, NY, NY) 719 p.