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Results:
Figure 2 shows the absorption spectra of heated and unheated chloroplasts which look quite similar. Figure 3 is a representation of heated and unheated chloroplasts as viewed from prepared slides, it can be seen that there are many more visible chloroplasts in the unheated chloroplast solution.
Discussion:
What did the absorbance spectra teach you about what was going on with the heated chloroplasts.
What did the microscopic evidence show about heating the chloroplasts?
You are scientific detectives trying to figure out what each treatment shows. What was the purpose of doing the experiment in the absence of light? What was the purpose of doing the experiment in the absence of chloroplasts? What did these two treatments show? What types of controls are each of these? What does the literature say about what effects the various treatments could have? Include what each rate/chloroplast showed both for DCPIP reduction and O2 evolution. Answer these questions from a molecular perspective as much as possible and use the literature to support or refute your findings. If your results differ from what was expected based on the literature, explain why. List sources of error, which are those errors inherent in the equipment used or experimental design. Include anything else that you think is relevant.
Chloroplasts are large organelles found mainly in leaf cells which are responsible for carrying out photosynthesis in plant cells.1They are lens shaped (which can be seen in figure 3) and contain three membranes, including a thylakoid membrane which is believed to be a single sheet that forms many tiny, interconnected flattened structures called the thylakoids (which are generally arranged in stacks called grana1.) Photosynthesis is the process of converting light energy into chemical energy through a series of reactions, many of which are light dependant.
The first step in photosynthesis is the absorption of light by chlorophylls which are attached to proteins in the thlyakoid membrane, chlorophyll is also what gives plants their characteristic green colour. The energy absorbed is used to remove electrons from a donor, H2O in green plants, forming oxygen and hydrogen ions. The electrons are then transferred to a primary electron acceptor, a quinone often designated as Q. The reduction takes place in photosystem II, which is a multiprotein complex driven by a wavelength of light less than 680nm1. In our experiment test tube 6 (which included the dye(DCPIP), STN buffer, and chloroplast solution) was not exposed to light, this resulted in no DCPIP reduction or O2 evolution because there was no light to for the chlorophylls to absorb and remove electrons from H2O. Test tube 7 which was used as a control and exposed to light did not contain any chloroplasts, only the dye and STN buffer, this test tube also showed no DCPIP or O2 evolution, which strengthens the assumption that it is the chloroplasts which are responsible for the O2 evolution in the other test tubes1.
The hill reaction which basically sums up the first step of photosynthesis is as follows 2 H2O + 2 A + (light, chloroplasts) → 2 AH2 + O2 , where A is the electron acceptor, in nature it’s usually NAD+, in this experiment we used DCPIP. It basically shows that with the presence of an electron acceptor, and light, chloroplasts can oxidize H2O to O2, and reduce the electron acceptor. Out of all the test tubes, test tube 2 which included the dye, STN buffer, and chloroplast solution served as a control and is the most similar to naturally occuring photosynthesis, with DCPIP reduction of 5.0377 x 10-12 per chloroplast and O2 evolution of 2.5189 x 10-12.
In the second step of photosynthesis electrons move from Q through a series of electron carriers until they reach the ultimate electron acceptor, in nature it’s usually NADP+, reducing it to NADPH1. In our experiment the ultimate electron acceptor was DCPIP, which was reduced to DCPIPH2, this occurs in a complex referred to as photosystem I which is driven by a wavelength of light less than 700nm. After this there is a generation of a proton-motive force, which leads to the synthesis of ATP and carbon fixation (conversion of CO2 into carbohydrates1.)
In test tube 3 we had the dye, STN buffer, chloroplast solution and DCMU. The effect of DCMU (which is a herbicide) was the inhibiton of photosynthesis, resulting in no DCPIP reduction or O2 evolution, this matches literary findings (Good, 1961). The effect of DCMU on the hill reaction is it interferes with the mechanism of water oxidation, which is what leads to the production of O2, and what drives the entire process of photosynthesis. How it does this is by blocking the Q(primary electron acceptor) binding site of ps II, if Q cannot accept any electrons then the entire process of shuttling the electrons to the ultimate electron acceptor, whether NAD+ or DCPIP, is halted, which in effect stops photosynthesis3. When photosynthesis in plants is inhibited they cannot produce the carbohydrates they need in order to survive and will die, so it makes perfect sense that adding a herbicide (plant killer) to the test tube would inhibit photosynthesis.
Test tube 4 contained dye, STN buffer, chloroplast solution, and the respiratory inhibitor sodium azide (NaN3.) Of all the test tubes this one had the highest rate of DCPIP reduction per chloroplast at 7.5105 x 10-12. There was no O2 evolution however, because sodium azide inhibits the transfer of electrons from cytochrome a3 to O25. Instead the electrons are picked up by other molecules such as DCPIP which explains why it has such a high reduction rate5. This means photosynthesis is unable to continue so sodium azide is also an inhibitor of photosynthesis, it just does it in a different way than DCMU.
Test tube 5 which contained the dye, STN buffer, and heated chloroplast solution, the DCPIP reduction per chloroplast was 1.83 x 10-13 and O2 evolution was 9.16 x 10-14, signifigantly smaller than the other test tubes which showed DCPIP reduction. This is because the chloroplast enzyme system is heat-senstive and slowly inactivated at room temperature, which is why we had to keep the chloroplast solutions on ice for the entire lab.
References:
1.textbook
2. Good, Norman E.. "Inhibitors of the Hill reaction." Plant Physiology 36(1961): 788-790.
3. FEBS Letters (1986) 205, 269-274
4.Maison-Peteri, B. "Effects of sodium azide on photosystem II of Chlorella pyrenoidosa.." Biochem
Biophys Acta. (1977): 459.
5. Bregman Allyn (1990) Laboratory Investigations in Cell & Molecular Biology (3rd ed),Wiley, NY
Figure 2 shows the absorption spectra of heated and unheated chloroplasts which look quite similar. Figure 3 is a representation of heated and unheated chloroplasts as viewed from prepared slides, it can be seen that there are many more visible chloroplasts in the unheated chloroplast solution.
Discussion:
What did the absorbance spectra teach you about what was going on with the heated chloroplasts.
What did the microscopic evidence show about heating the chloroplasts?
You are scientific detectives trying to figure out what each treatment shows. What was the purpose of doing the experiment in the absence of light? What was the purpose of doing the experiment in the absence of chloroplasts? What did these two treatments show? What types of controls are each of these? What does the literature say about what effects the various treatments could have? Include what each rate/chloroplast showed both for DCPIP reduction and O2 evolution. Answer these questions from a molecular perspective as much as possible and use the literature to support or refute your findings. If your results differ from what was expected based on the literature, explain why. List sources of error, which are those errors inherent in the equipment used or experimental design. Include anything else that you think is relevant.
Chloroplasts are large organelles found mainly in leaf cells which are responsible for carrying out photosynthesis in plant cells.1They are lens shaped (which can be seen in figure 3) and contain three membranes, including a thylakoid membrane which is believed to be a single sheet that forms many tiny, interconnected flattened structures called the thylakoids (which are generally arranged in stacks called grana1.) Photosynthesis is the process of converting light energy into chemical energy through a series of reactions, many of which are light dependant.
The first step in photosynthesis is the absorption of light by chlorophylls which are attached to proteins in the thlyakoid membrane, chlorophyll is also what gives plants their characteristic green colour. The energy absorbed is used to remove electrons from a donor, H2O in green plants, forming oxygen and hydrogen ions. The electrons are then transferred to a primary electron acceptor, a quinone often designated as Q. The reduction takes place in photosystem II, which is a multiprotein complex driven by a wavelength of light less than 680nm1. In our experiment test tube 6 (which included the dye(DCPIP), STN buffer, and chloroplast solution) was not exposed to light, this resulted in no DCPIP reduction or O2 evolution because there was no light to for the chlorophylls to absorb and remove electrons from H2O. Test tube 7 which was used as a control and exposed to light did not contain any chloroplasts, only the dye and STN buffer, this test tube also showed no DCPIP or O2 evolution, which strengthens the assumption that it is the chloroplasts which are responsible for the O2 evolution in the other test tubes1.
The hill reaction which basically sums up the first step of photosynthesis is as follows 2 H2O + 2 A + (light, chloroplasts) → 2 AH2 + O2 , where A is the electron acceptor, in nature it’s usually NAD+, in this experiment we used DCPIP. It basically shows that with the presence of an electron acceptor, and light, chloroplasts can oxidize H2O to O2, and reduce the electron acceptor. Out of all the test tubes, test tube 2 which included the dye, STN buffer, and chloroplast solution served as a control and is the most similar to naturally occuring photosynthesis, with DCPIP reduction of 5.0377 x 10-12 per chloroplast and O2 evolution of 2.5189 x 10-12.
In the second step of photosynthesis electrons move from Q through a series of electron carriers until they reach the ultimate electron acceptor, in nature it’s usually NADP+, reducing it to NADPH1. In our experiment the ultimate electron acceptor was DCPIP, which was reduced to DCPIPH2, this occurs in a complex referred to as photosystem I which is driven by a wavelength of light less than 700nm. After this there is a generation of a proton-motive force, which leads to the synthesis of ATP and carbon fixation (conversion of CO2 into carbohydrates1.)
In test tube 3 we had the dye, STN buffer, chloroplast solution and DCMU. The effect of DCMU (which is a herbicide) was the inhibiton of photosynthesis, resulting in no DCPIP reduction or O2 evolution, this matches literary findings (Good, 1961). The effect of DCMU on the hill reaction is it interferes with the mechanism of water oxidation, which is what leads to the production of O2, and what drives the entire process of photosynthesis. How it does this is by blocking the Q(primary electron acceptor) binding site of ps II, if Q cannot accept any electrons then the entire process of shuttling the electrons to the ultimate electron acceptor, whether NAD+ or DCPIP, is halted, which in effect stops photosynthesis3. When photosynthesis in plants is inhibited they cannot produce the carbohydrates they need in order to survive and will die, so it makes perfect sense that adding a herbicide (plant killer) to the test tube would inhibit photosynthesis.
Test tube 4 contained dye, STN buffer, chloroplast solution, and the respiratory inhibitor sodium azide (NaN3.) Of all the test tubes this one had the highest rate of DCPIP reduction per chloroplast at 7.5105 x 10-12. There was no O2 evolution however, because sodium azide inhibits the transfer of electrons from cytochrome a3 to O25. Instead the electrons are picked up by other molecules such as DCPIP which explains why it has such a high reduction rate5. This means photosynthesis is unable to continue so sodium azide is also an inhibitor of photosynthesis, it just does it in a different way than DCMU.
Test tube 5 which contained the dye, STN buffer, and heated chloroplast solution, the DCPIP reduction per chloroplast was 1.83 x 10-13 and O2 evolution was 9.16 x 10-14, signifigantly smaller than the other test tubes which showed DCPIP reduction. This is because the chloroplast enzyme system is heat-senstive and slowly inactivated at room temperature, which is why we had to keep the chloroplast solutions on ice for the entire lab.
References:
1.textbook
2. Good, Norman E.. "Inhibitors of the Hill reaction." Plant Physiology 36(1961): 788-790.
3. FEBS Letters (1986) 205, 269-274
4.Maison-Peteri, B. "Effects of sodium azide on photosystem II of Chlorella pyrenoidosa.." Biochem
Biophys Acta. (1977): 459.
5. Bregman Allyn (1990) Laboratory Investigations in Cell & Molecular Biology (3rd ed),Wiley, NY