Introduction
Rubisco, one of the most important enzymes in photosynthesis, is hypothesized to be the most abundant protein on Earth (Gurevitch et al. 2002). This enzyme is so important to photosynthesis that it makes up 50% of the protein in a chloroplast (Lodish et al. 2003). Rubisco consists of eight identical large subunits and eight identical small subunits. The large subunit mRNA is transcribed from DNA (rbcl gene) in the chloroplast, while the small subunit mRNA is transcribed in the nucleus. The enzyme plays a crucial role in photosynthesis by catalyzing a reaction to fix CO2 to ribulose bisphosphate, a 5-carbon molecule. The fixation of CO2 is the first step of the Calvin cycle through which a 3-carbon sugar is made to eventually create a storable energy for plants. Rubisco has an affinity for both CO2 and O2; consequently, both molecules are in constant competition for rubisco's binding site. This poses a problem for plants because when O2 is fixated to ribulose bisphosphate, photorespiration occurs instead of photosynthesis; this reduces the amount of sugar made by the plant and causes the loss of ATP.
In warm environments, photorespiration poses a large problem. Stomata must be closed during the day to prevent excessive water loss; consequently, CO2 levels inside mesophyll cells drop drastically. Lower CO2 levels allow O2 to bind to rubisco at higher rates, thus photorespiration increases while photosynthesis decreases and less glucose is made as a result. C4 plants, unlike C3 plants, have adapted to overcome this problem.
In C3 plants, CO2 is fixated by rubisco in mesophyll cells where transpiration and respiration occur through stomata. However, C4 plants evolved metabolic and anatomical adaptations to separate the activities of the stomata from the Calvin cycle. C4 plants have adapted by compartmentalizing the Calvin cycle into bundle sheath cells and carbon fixation into mesophyll cells. PEP carboxylase is the key enzyme that allows this process to occur in C4 plants. PEP carboxylase has a much higher affinity for CO2 than O2, thus allowing it to bind to CO2 at very low concentrations. After binding to CO2 in the mesophyll cell, PEP carboxylase transfers it into the bundle sheath cell through a series of processes. This creates a high concentration of CO2 in the bundle sheath cell. Therefore, C4 plants have a higher concentration of CO2 where the Calvin cycle occurs than do C3 plants. This not only allows C4 plants to prevent water loss better than C3 plants by allowing the stomata to be closed more often, but it also prevents high rates of photorespiration.
In this study we are particularly interested in the differences between the quantities of the rubisco large subunit and its counterpart DNA, the rbcl gene, in photosynthetic cells of C3 and C4 plants. The amount of rubisco in cells is controlled by transcription, translation, and protein degradation. Ku et al. (1979) demonstrated that rubisco in three species of C3 plants (Nicotiana tabacum, Solanum tuberosum, Triticum aestivum) makes up 25-60% of proteins in the leaf and only 8-23% of proteins in three species of C4 plants (Panicum miliaceum, Panicum texanum, Zea mays). In the same study, the researchers found that C4 plants have higher rates of photosynthesis than C3 plants because CO2 concentration is not a limiting factor in this process. Schmitt and Edwards (1981) demonstrated that maize, a C4 plant, showed better use of rubisco than wheat or rice which are C3 plants. They concluded that C4 plants have a better nitrogen use efficiency (amount of CO2 fixed per unit of N) than C3 plants. This was supported by experiments performed by Sage et al. (1987). Another study revealed that rubisco in C4 plants displayed a higher catalytic efficiency than C3 plants in warm climates (Sage 2002).
Our hypothesis will test the idea that there will be less rubisco large subunit protein and rbcl gene in photosynthetic cells of C4 plants compared to C3 plants. This idea is based on previous research and the fact that rubisco in C4 plants operates at higher concentrations of CO2 than C3 plants. This reduces the amount of competition for the binding site of rubisco by O2. Therefore, the high concentration of CO2 reduces the level of rubisco required to create the sugars needed for plant survival. To test this hypothesis, we will compare the quantities of the rubisco large subunit and the rbcl gene of two C3 plants, Poa pratensis (Kentucky bluegrass) and Acer saccharum (sugar maple), and two C4 plants, Digitaria sanguinalis (crabgrass) and Zea mays (maize).