Introduction Methods Results Discussion Literature Cited
Discussion
Plants with C3(Impatiens walleriana), C4(Zea mays), and CAM(Kalanchoe prolifera) carbon fixation cycles were selected for comparing Rubisco expression. The species used were chosen based on this characteristic and availability.
Primers specific for the RBCL gene were designed and used in Real Time PCR to quantify the amount of DNA present in each plant sample. Each sample was also separated by size using agarose gel electrophoresis to confirm that the correct product was created in the reaction. Bands were observed around 200bp for every sample on the gel, meaning that the reaction worked correctly and the RBCL gene was amplified in every sample. The most conclusive Real Time PCR results were found by using 50ng of template DNA. The Ct values for each sample indicated that the highest concentration of RBCL DNA was present in the C3 plant and lowest in the CAM samples. Ct values for C4 fell between the C3 and CAM values. This indicates that the highest level of Rubisco was being produced in the C3 plant. Contamination was not observed in the negative control and Tm values also indicate that DNA was amplified. Tm values were read at cycle 7 and ideally needed to be around 84 degrees Celsius to indicate the presence of the correct sized product. All samples had a Tm value close to this standard. The RBCL gene is contained in plastid DNA meaning that these results measure the amount of chloroplasts, leucoplasts, and chromoplasts that were present in our plant tissue.
SDS-PAGE gels were used to separate isolated proteins and identify the Rubsico large subunit protein. The first gel was just used to separate proteins by size and stained. Bands were observed around 55,000 kDa for each sample and this matches the size of the Rubisco large subunit. Bands for the C4 and CAM samples were much fainter than the C3 band indicating that less protein was probably present. Another SDS gel was used to perform a Western blot. The control band was not present, so the antibodies could not be concluded that they did indeed bind to the membrane correctly. Bands were present in both the C3 and C4 lanes, and in one of the CAM lanes. The other CAM sample did not have a band. The highest protein concentration was also found in the C3 sample and the lowest in the CAM samples. According to the standard values found using BSA concentrations the C3 plant had a much higher protein concentration that either the C4 or CAM plants. The second CAM sample was probably not detected in the Western blot because it contained the least amount of protein. This coincides with the higher Ct value found in the Real Time PCR amplification. The least amount of Rubisco was being produced in the CAM plant and the most in the C3 plant.
Based on these results, we reject our original hypothesis. Both the DNA and protein results indicate that C3 plants express higher levels of Rubisco than C4 or CAM plants. The carbon fixation cycles of the C4 and CAM are apparently not as dependent on Rubisco for carbon fixation. Since the C4 and CAM photosynthetic pathways do not have the same amount of carbon available, they have developed carbon-concentrating mechanisms. These mechanisms play a selective role in the expression of Rubisco. These mechanisms also utilize separate pathways that do not require Rubisco in order to fix carbon. Therefore, these photosynthetic pathways require less Rubisco to fix carbon regardless of the environment (Keeley). The difference in photosynthetic pathways is the reason that the C3 sample contained more Rubisco in their chloroplasts than the C4 and CAM samples. Having a more efficient photosynthetic pathway probably contributes to a decreased concentration of Rubisco at certain times because carbon is only fixed in C4/CAM plants when Rubisco concentration is high during the daytime. Rubisco is always present in C3 plants, so it leads us to believe that this protein is constantly around at high concentration due to C3's inefficient carbon fixation pathway.
Further research could be done to determine the effect of these photosynthetic pathways on Rubisco expression. A comparison of different C4 and CAM plants could determine whether it is in fact the photosynthetic pathways or whether it is the environment that drives Rubisco expression. Analysis of C4 and CAM plant Rubisco concentration could be done using different species and detecting the amount of Rubisco that is being expressed during the day and at night. Additionally, C3 plants from different environments could be compared to one another to further investigate the effects of environment on Rubisco expression. Environmental changes we could look at would be if expression levels vary in warmer or colder climates as well as sunny or dark climates.