W
|
WELCOME |
|
Web
Site for Sara
Wacker-Katie Meinhover "......if
you have any questions about our research paper please |
|
|
The
following research paper was prepared by Katie and Sara. If you have
questions or suggestions please
Introduction:
The growth of the world’s population has historically been accomplished by
technological innovation. Great advances in farming techniques and tools have
kept pace with population explosions without causing large numbers of people to
go hungry. Recently the world's population has reached the 6 billion mark and
out of those people, 800 million suffer from hunger and malnutrition (Food and
Agricultural Organization of the
Understanding the structure and processes of plants is crucial to improving agricultural production worldwide. The internal structure of the leaf is complex. In order to understand photosynthetic processes, it is vital to first comprehend leaf structure. In the base of the leaf, there are small openings called stomates that regulate vital elements such as carbon dioxide and water. Once these elements enter through the stoma they reach a layer called the mesophyll. Inside the mesophyll cells, there are organelles that are called chloroplasts. The chloroplasts in the cells are surrounded with a protein fluid called the cytoplasm. The internal structure of a chloroplast consists of groups of thylakoids stacked on top of each other (Raven & Johnson, 1996). These stacks are then classified as grana. Thylakoids contain a green substance called chlorophyll that is used to trap light energy for photosynthesis.
Photosynthesis is the process by which plants use energy from the sun's rays to convert carbon dioxide and water into carbohydrate energy using the chlorophyll in the chloroplast of the plant's leaves. (Raven & Johnson, 1996) Photosynthesis consists of two main reactions. One occurs in the light and the other takes place after the light reaction. In the basic light reaction, the sun's rays (or another source of light energy) is absorbed by chlorophyll, an energy carrier, that will become energized. This energized chlorophyll will then supply energy to split a molecule of water into two hydrogens and oxygen. The oxygen is released into the atmosphere, while the two atoms of hydrogen are trapped by nicotinamide adenine dinucleotide phosphate (NADP), which will be used in the Calvin Cycle. During the Calvin Cycle, carbon dioxide is combined with ribulose-1,5-biphosphate (RuBP), a five-carbon sugar found in the chloroplast. This reaction is catalyzed by rubisco (RuBP carboxylase). Together these form a very unstable six-carbon sugar, which immediately divides into two molecules of phosphoglyceric acid (PGA). These three-carbon compounds react with the two atoms of hydrogen that were brought from the light reaction by NADP, forming water which is released as a by-product. Also, phosphoglyceral aldehyde (PGAL) is formed; this can be used directly by the cell as a nutrient or converted into glucose (Otto,Towle, & Bradley, 1981). This most widely-known type of photosynthesis is referred to as C3 photosynthesis. The name comes from the three-carbon compound, PGA, around which the reaction is centered.
A second form of photosynthesis, called C4 photosynthesis, was identified by Marshall D. Hatch, a plant physiologist and biochemist (Hatch, 1992). This photosynthetic pathway has a four-carbon compound, malic acid, which takes the place of PGA. Many scientists believe that the C4 pathway has evolved from C3 plants because of dramatic temperature changes and a large percentage of oxygen in the earth's atmosphere. This pathway begins with CO2 entering through the stomata of a plant's leaf and into the mesophyll cells. Once in the mesophyll, the carbon dioxide combines with phosphoenolpyruvic acid (PEP) to produce oxaloacetic acid (OAA). OAA is a four-carbon acid that quickly moves into an adjacent bundle sheath cell. There the acid is split into a carbon dioxide and a three-carbon compound that moves back into the mesophyll to continue the
process. The carbon dioxide combines with RuBP, triggered by rubisco. (Uno, Storey, & Moore, 2001)
The main difference in these two photosynthetic processes is that in the C4 pathway only carbon dioxide can combine with the RuBP. In the C3 pathway, rubisco may allow the RuBP to combine with carbon dioxide or oxygen, whichever is more readily available. When RuBP combines with the oxygen, photorespiration occurs, which does not produce any ATP (adenosine triphosphate-a high energy molecule). Photorespiration, or the process of rubisco fixating O2, is a useless waste of energy and time. The amount of energy that photorespiration produces is less than the amount that it uses. Forty percent of the energy produced during C3 photosynthesis is estimated to be lost during photorespiration (Tootill, 1991). On the other hand, in the C4 pathway, CO2 is fed directly to RuBP and is the only element rubisco is allowed to fixate. In the long run, this enables energy to be produced at a quicker rate (Uno, Storey & Moore, 2001). However, the C4 pathway is not always more efficient. Environmental temperatures and light energy greatly affect how useful the evolved pathway is. An ATP molecule is used to move the PEP enzyme back into the stomata. When the average temperature is below 28 degrees Celsius and when there is less light, the C3 cycle occurs less frequently and the chance for photorespiration decreases. Thus, it is not worthwhile to expend an ATP molecule in the evolved C4 pathway (Encyclopedia Britannica, 1999).
While working with the C4 pathway, Hatch and Slack found a very unique enzyme, pyruvate orthophosphate dikinase (PPDK). It was discovered that this enzyme catalyzes a reaction with ATP and Pi and produces PEP, pyrophosphate (PPi), and AMP (Dave Kubien, email to author, Jan 8, 2001). Thus, PPDK is vital to C4 plants because it creates PEP. Another remarkable characteristic of this enzyme is that its activity is regulated between light and dark. When phosphorylation of PPDK occurs, the PPDK enzyme is turned off by a bifunctional regulatory protein, which deactivates photosynthesis (Sheriff et al, 1998). This phosphate is then taken away as a direct light source appears.
PPDK, first discovered in C4 plants, has recently been identified in plants that use the C3 pathway. J. Sheen at the Department of Genetics in Harvard Medical School (1991) made a discovery of a transit amino acid peptide gene involved in the transfer of PPDK within the cytoplasm. She cloned the transit peptide gene and discovered that it was nonphotosynthetic. This is significant because the specific function of PPDK in C3 plants is also unknown. Assuming that C4 plants evolved from C3 plants, Sheen theorized that the function of the nonphotosynthetic PPDK of the C4 plants was the same as the PPDK in C3 plants. Then, in 1997, Imaizumi cloned the PPDK gene in rice, which is a C3 plant. They discovered that there were two different strands of PPDK genes: one that was supposed to be located in the cytoplasm, and the other should have been found in the chloroplast (Imaizumi et al, 1997). The background sources that we investigated did not clarify the location of PPDK in the chloroplasts of C3 plants.
The agricultural revolution of the 1700’s contributed to an era of population growth and development. Today, an enlarging world population continues to demand an elevated food production. Agricultural science is now entering a new frontier where food can be genetically engineered and produced more efficiently. Understanding the connection between C3 and C4 photosynthetic pathways is a step into this new frontier. Once major plant enzymes, like PPDK, are understood, the application of genetics in food production becomes increasingly useful in feeding the hungry. It is the purpose of this study to determine if pyruvate, orthophosphate dikinase is located in the chloroplasts C3 plants.
Hypothesis:
PPDK is located in the chloroplasts of C4 plants and is a major enzyme used in photosynthesis. In 1991, Sheen reported that PPDK was located in C3 plants, but its role in the plant was not established. Another study done on a C3 plant, rice, showed that PPDK had the necessary genetic instructions to be targeted in the chloroplasts (Imaizumi et al, 1997). Based on this discovery, we believe PPDK will be located within the chloroplast of C3 plants.
Methods:
The procedure began with chloroplast isolation. Using a simple household blender, spinach leaves were ground up; regular cheesecloth was then used to strain the liquid. The liquid was then centrifuged to separate the broken and intact chloroplasts from the rest of the liquid. The pellet was re-suspended and layered on top of a percoll cushion to separate the lysed chloroplasts from the intact ones. This was then centrifuged in a swing bucket centrifuge to collect only the intact chloroplasts.
The chloroplasts were then lysed with a hypotonic buffer before we began the process of ammonium sulfate fractionation to interact with the salt bonds that hold the proteins together. Ammonium sulfate was added until it equaled 35% of our solution. The mixture was centrifuged in order to remove unnecessary proteins. Keeping only the liquid, this process was repeated until the solution contained 55% ammonium sulfate. Upon centrifuging, the remaining pellet contained all the chloroplast proteins that precipitate out between 35-55% ammonium sulfate, which should include PPDK.
The third step after ammonium sulfate fractionation was protein analysis. In this method, a
computerized spectrophotometer was used to read the concentration of different proteins. At this
point, there were five different samples collected from previous experiments to analyze. Comparing and analyzing each sample distinguished how much protein was needed in loading the electrophoresis SDS-page gel.
Once identified, the protein levels of the samples were ready to be loaded. To activate the proteins, they were heated. The columns were arranged in an order to be loaded. The next step was to load the proteins into the gel using a pipette. When this was completed, the Western Blot- electrophoresis- station was set up. After an hour, the gel was ready to be transferred to the nitro-cellulose membrane. The next step was forming a transblotting sandwich where the gel and membrane were next to each other. The sandwich was then soaked in buffer with an electrical current that transferred the proteins onto the membrane.
After transblotting the proteins, the membrane was soaked in the primary antibody solution for either PPDK or PEPcarboxylase. The primary antibody builds upon the protein that it is designated for, in this case, PPDK or PEP carboxylase. The membrane was incubated over night and was then ready to be washed in buffer, which removed the primary antibody. Soaking the membrane in secondary antibody, which would attach to the primary antibody, continued this process. Then the membrane was washed again and immersed in Pierce Alk phop reagent, which acted as a developer for the secondary antibody. Soon after, bands appeared. These bands were then analyzed by visual observation and comparison to other membrane samples to determine if PPDK was in the chloroplast.
Data:
Trial 1:
The first gel shown in Figure 1 (F1) contains proteins collected from one leaf sample and one chloroplast sample. In addition, a faba bean chloroplast sample collected by Dr. Christopher Chastain was also run on this gel. This sample was transblotted with PEPc (PEPcarboxylase) antibody. Since PEPc is only found in the cytoplasm of the plant leaf, the absence of bands in lanes one and two indicate that chloroplasts have been isolated. Figure 2 (F2) is a new gel loaded with the same samples as F1. This gel was transblotted onto a membrane that was soaked in PPDK antibody. The presence of bands in lanes one and two is evidence that PPDK is present in the chloroplasts.
Figure 1: Western Blot probed in PEPcarboxylase
Figure 2: Western Blot probed in PPDK Antibody
Trial 2:
The second electrophoresis trial included two spinach leaf samples and three spinach chloroplast samples. Figure 3 (F3) was probed with PEPc antibody. In lanes one and two PEPc is visible while lanes three, four and five did not show the presence of PEPc. This verifies that the three isolated chloroplasts samples did not contain anything except chloroplasts. Figure 4 (F4) has the same samples as F3, but was soaked with PPDK antibody. This figure shows that in all lanes PPDK is present.
Figure
3: Western Blot probed with PEPc antibody
Figure 4: Western Blot probed with PPDK antibody
Conclusion:
Our hypothesis that PPDK would be present within C3 chloroplasts was verified through our research. In figure F2, PPDK is found in isolated spinach and faba bean chloroplasts, both are C3 plants. F4 shows that PPDK was also located in three different isolated chloroplast samples. Our data clearly shows that pyruvate, orthophosphate dikinase is indeed located inside the chloroplasts of C3 plants.
Discussion:
In this investigation, steps were taken to ensure data collected would be reliable. For example, an extra step in chloroplast isolation separated the lysed from the intact chloroplasts. Most scientists believe that chloroplast isolation with out this security step is 99% accurate (C. Chastain, personal interview, 1-28-01). In addition, ammonium sulfate was used to selectively precipitate out unwanted proteins, further concentrating PPDK from the chloroplast samples. These steps coupled with consistent, replicated data indicate that the information collected is valid. All of the data collected with the Western Blot technique confirmed the presence of PPDK in C3 plant chloroplasts, as detected by highly specific antibody for PPDK.
Locating PPDK in the chloroplast of C3 plants is an essential step to understanding this important enzyme. PPDK’s role in phosphorylation and in light and dark regulation still needs to be clarified in C3 plants. It is known that PPDK within C4 plants becomes phosphorylated in the dark and dephosphorylated in the light. Further research may include studying if PPDK behaves similarly in C3 plants. From there, it may be beneficial to investigate why phosphorylation of PPDK occurs in C3 plants even though PPDK is not linked to C3 photosynthesis. This will provide an important clue as to what PPDK does in C3 plants. Why does it need to be on in the light and off in the dark; why does it need to be so closely linked to the photosynthetic process? Is this why most of the PPDK is located in the chloroplast to begin with?
Every time a new discovery
is made, it affects someone, whether in the scientific world or outside of it.
Locating PPDK in C3 chloroplasts is the end of one study, and the beginning of
a new one. A scientist’s role in science is to uncover tiny pieces of a puzzle,
like ours. With work, other pieces of the puzzle will be revealed to compose a
larger picture. Scientists Sheen and Imaizumi
predicted that pyruvate, orthophosphate dikinase could be found in the cytoplasm and the
chloroplast of plants that use the C3 photosynthetic pathway. This prediction,
however, had not been tested until now. This research is breaking ground for
further studies. The study we conducted will open the door and shed light on
new photosynthetic discoveries. .
Literature Cited
Economic and Social Council Meets to Discuss ‘Eliminating Hunger in the New Millenium.’ (Oct. 30, 2000) Food and Agricultural Organization of the United States. Press Release ECOSOC/5935.
Hatch, M.D. (1992). C4 photosynthesis: an unlikely process full of surprises. Plant Cell Physiology. 4: 333-342.
Imaizumi, N., Ku, M.S.B., Isihara, K., Samejima, M., Kaneko, S., & Matsuoka, M. (1997). Characterization of the gene for pyruvate, orthophosphate dikinase from rice, a C3 plant, and comparison of structure and expression between C3 and C4 genes for this protein. Plant Molecular Biology 34: 701-716.
Otto, J., Towle, A., & Bradley, J. (1981). Modern Biology. New York, NY: Holt, Rinehart, and Winston, Publishers.
Raven, P., & Johnson, G. (1996). Biology. Dubuque, IA: Times Mirror Higher Education Group, Inc.
Sheen, J. (1991). Molecular mechanisms underlying the differential expression of maize pyruvate, orthophosphate dikinase genes. Plant Cell 3: 225-345.
Sheriff, A., Meyer, H., Riedel, E., Schmitt, J. M., & Lapke, C. (1998). The influence of plant pyruvate, orthophosphate dikinase on a C3 plant with respect to the intracellular location of the enzyme. Plant Science 136: 43-57.
Specific Variations in
Photosynthesis. (1999-2000). Retrieved
Tootill, E. (1981). The
Facts on File: Dictionary of Biology.
Uno, G., Storey, R., & Moore, R. (2001). Principles
of Botany.