Discussion
Protein Analysis
After protein concentration calculations were calculated in Table1 for each tissue sample, it was found that petals had the highest concentration of protein for both plants, followed by leaves and stems. A reason for the petals’ significantly higher protein concentration may be due to pigments that absorbed more light during the spectrophotometer readings. The petals were a violet color, resulting in a darker solution in the cuvettes. The leaves samples also may have pigments that could have interfered with absorption of light. These darker solutions could have caused a higher absorbance reading, skewing protein calculation results.
A SDS-PAGE was performed in order to separate proteins by their charge and size. If Rubisco was present in our protein solution, the gel would have shown a protein band between the green marker at 85kDa and violet market at 45.7kDa indicating Rubisco. However, the SDS gel did not show any significant protein bands, even for the positive control in Figure2. This may have resulted because there were not enough protein sample used to load the gel to show a significant band, since pigments may have resulted in over-calculation of the protein concentrations. An absent of bands could also have been a result that the protease inhibitors didn’t perform correctly; proteases may have degraded the proteins before they were loaded into the gel. But since the positive control didn't show either, source of error was probably due to an insufficient amount of protein, and not protease inhibitors.
The Western blot was performed to determine whether or not Rubisco protein was present in plant protein samples. The results showed that there were no Rubsico bands present for petals in both plants, seen in Lanes 3 and 8 of Figure 3. This concludes that there is not a significant amount of Rubisco in petals, compared to other plant parts. Rubisco protein in stems in Dianthus and Chrysanthemum were detected by antibodies and apparent by the bands in Lanes 1 and 6 of Figure 3. These bands were slightly darker than the others, concluding that Rubisco protein was more abundant in stems. The Dianthus leaves were detected to have the Rubisco protein, but the leaves of the Chrysanthemum did not show any signs of Rubisco present. The band for Chrysanthemum leaf protein may not have shown due to errors in loading the gel or that there was not enough isolation of cells during the experiment.
In Figure 3, since stems of both plants detected Rubisco protein and also had darker bands in the Western Blot, it was concluded that stems did have a greater amount of Rubisco protein when compared to leaves and petals. This was against the hypothesis that leaves would have more protein since photosynthesis is mainly done through leaves. The stem’s photosynthetic properties was underestimated when formulating a hypothesis. Studies have been conducted that have actually shown that stem photosynthesis is qualitatively similar to leaf photosynthesis, and that photosynthetic activity of stems in the plant Cericidium floridium is comparable to that of leaves when computed on a rate per unit area (Adams et al, 1967). There are many plants that have no leaves at all and therefore must get all its photosynthetic nutrients from stems. For example, the plant Fouquieria splendens is leafless most of the year because of drought so stems are essential for photosynthesis in order to survive (Nedoff et al., 1985). From the results of this experiment, stems must be an important contributor in generating the photosynthetic nutrients for both plants.
DNA Analysis
In addition to Rubisco protein, Rubisco DNA can also be found in chromoplasts, leucoplasts, and chloroplasts. These plastids are found throughout the plant, therefore it was hypothesized that there would be relatively equal amounts of Rubisco DNA within leaves, petals, and stems. Again, DNA was first isolated from the different plant tissues and concentrations of DNA for each plant tissue was calculated. Results in Table 4 showed that Chrysanthemum petals had a significantly higher concentration in comparison to its leaves and stems, and the leaves contained the least amount of DNA. Again, it is possible that the petals’ significantly higher calculated concentration was due to pigments, which can absorb more light at 260nm and 280nm in the spectrophotometer. On the other hand, Dianthus stems had a greater DNA concentration and petals had the least for this plant.
Real-Time Polymerase Chain Reaction (PCR) was performed in order to determine which tissue samples had the highest concentration of Rubisco DNA. This was determined through C(T) values, the cycle number at which each sample reaches a set threshold. The lower the C(T) values, the greater amount of Rubisco DNA was expected to be in the sample. It was surprising to see that the two trials had different outcomes while using the same amount of Rubisco DNA. Source of error was probably due to pipetting errors while transferring tissue samples into the tube for PCR.
In Chrysanthemum, stems had the lowest C(T) value and leaves had the highest C(T) value for both trials of PCR. This data shows that there was more Rubisco DNA in stems than leaves for Chrysanthemum, which didn’t support the hypothesis that Rubisco DNA was evenly distributed in the plant. Again, the stems may have the same photosynthetic capabilities as leaves do which could have resulted in the higher Rubisco DNA in this experiment. The low C(T) value for Chrysanthemum leaves could have been caused by collecting too few cells in the start of the experiment when isolating the Rubisco DNA from the leaves. The PCR data for Dianthus showed that leaves had the lowest C(T) value, petals had no C(T) value, and stems had the highest value in both PCR Trials. This didn’t support the hypothesis that Rubisco DNA is evenly distributed throughout the plant. In this case, leaves had more Rubisco DNA, which shows that this part of the plant is an important factor of photosynthesis. It was surprising that petals didn’t have a C(T) value from the PCR. Either petals did not have any Rubisco DNA or there were human errors during the processing of these petals.
To ensure that Rubisco DNA was present after PCR, an agarose gel was performed to determine whether Rubisco DNA was amplified. Results showed that DNA was present in leaves of both plants seen in Lanes 1 and 2 of Figure 10. Rubisco DNA was present in the Chrysanthemum petals seen in Lane 5 of Figure 10. DNA was not detected in Dianthus petals, which can explain why there wasn’t a C(T) value in the PCR analysis. There was also a light band of Rubisco DNA found in the “No DNA” lane of the gel seen in Lane 4 of Figure 10. As a negative control, it was expected that DNA would not be present in this lane. Since DNA was found, results could have been skewed for the data in the PCR analysis.
After analysis of all Rubisco DNA data, it can be concluded that the concentration of Rubisco DNA was greater in photosynthetic plant tissues, such as the stems and leaves. Photosynthetic tissues need more Rubisco protein, and therefore more Rubisco DNA. As to which photosynthetic tissue, stems or leaves, had the most Rubisco DNA could not be determined by this experiment. There may be other factors that can determine whether stems or leaves have a greater concentration of Rubisco DNA between the two species used in this experiment. The size and shape of the leaf could have contributed to the differences in amounts of DNA in the leaves of different species. Dianthus has longer slender leaves whereas Chrysanthemum has shorter, rounder leaves. Anatomy of the leaves could have an effect in cell size and shape since Dianthus leaves had the greatest concentration of DNA, unlike Chrysanthemum where stems had the highest concentration. Dianthus leaved may have more cells per area than Chrysanthemum leaves, resulting in more DNA.
Another factor that could have affected data is that Chrysanthemums are perennials whereas Dianthus are annuals. Since perennials must survive winter conditions, it is possible that they may have to store more nutrients, including DNA. Since one of the main functions of stems is storage of nutrients, this may be a reason why stems of Chrysanthemums have higher DNA amounts than the annual Dianthus.
To further this research, multiple trials will have to be conducted in order to draw better conclusions on whether leaves, stems, or petals have more Rubisco DNA and protein. Other parts of the plants can be tested as well, to see if the function correlates to amounts of Rubisco present. A further in depth study between perennials and annuals can be conducted to find if there are any differences in Rubisco DNA and protein between the two types of plants.