Discussion
Various factors contribute to inconsistencies of rubisco concentrations between different species of plants or even between different parts of the same plant (Sage 2002). One such factor is the way that certain plants fixate carbon dioxide in different climates. Although both contain chloroplasts and rubisco, C4 plants differ from C3 plants in that they compartmentalize available CO2 and consequently use rubisco to different degrees than C3 plants do (Schmitt and Edwards 1981). Our study focused on comparisons of the concentrations of the rubisco protein and its corresponding rbcl gene in the photosynthetic cells of C4 plants and C3 plants. We chose to compare tissues of the C3 and C4 grasses (P. pratensis and D. sanguinalis respectively) and the leaves of C3 and C4 non-grasses (A. saccharum and Z. mays respectively). Our results did not validate differences in rubisco protein concentrations. However, we observed differences in rubisco gene expression that support our hypothesis that the concentrations of rubisco and the rbcl gene would be greater in the C3 plants P. pratensis and Acer saccharum than C4 plants, D. sanguinalis, and Z. mays.
Although transcriptional regulation of rubisco exists, it cannot be expected to be a reliable predictor of rubisco expression by itself. Many factors, including variations of concentrations of chloroplasts localization between samples, must be taken into consideration when associating rbcl gene quantitation as a determinant for rubisco quantitation. Consequently, quantitation of the large subunit gene must be accompanied by a quantitation of the protein as well to verify any results.
To begin with, our agarose gel in Figure 6 showed us that, as expected, primers A and B worked properly. Both bound to our DNA and displayed bands of 300-500 and 200 bp respectively for our samples in lanes 1,2,4 and 5. Although we did not see any bands in our primer A blank in lane 3 as expected, we did see a 200 bp band for our blank with primer B. We determined that the band was a result of contamination with one of our tissue samples since it looked like our primer B products seen in lanes 4 and 5.
Given that both primers were specific only for the rbcl gene, it was possible to confidently perform Real-Time PCR and expect that only our rbcl gene would be quantitated. In RT-PCR, once a sybr green protein binds to double stranded DNA (dsDNA) it fluoresces after its electrons are excited by a laser. As a result, the fluorescence of sybr green detected reflects dsDNA concentrations. Ct values indicate the cycle at which DNA replication surpassed background levels and increased dramatically. Thus, the Ct value also corresponds to the relative concentration of double stranded DNA in any one sample, with lower Ct values representing larger rbcl gene concentrations.
The melting curve shown in Figure 3 was used to find that 78 °C was the optimal temperature to observe Ct values for primers A and B. The Tm for Primer A could easily be determined because we knew that it needed to lie somewhere in between the Tm of the primer dimers (76°C) and the Tm of the rbcl gene (82°C) at which only 50% of our DNA remained. Primer B did show a Tm for rbcl around 82 °C, but no Tm for primer dimers before it, which was in accord with the fact that no primer dimers were found in lane 6 of our agarose gel (Figure 6) either. Therefore, for Primer B 78 °C was still used as the Tm at which to observe Ct values.
When comparing the data for figure 5, it seems as though something went awry because the same products using primer A or B were larger in the 25 ng samples than in the 50 ng samples. We would expect that since the 50 ng samples had twice as much DNA loaded, there would be larger values than for the 25 ng samples. However, when looked at individually, some trends begin to appear. The data from Figure 5 indicates that using Primer B for 25 ng DNA samples, C3 plants contained lower Ct values, and consequently larger amounts of rbcl gene than C4 plants. For every other run (Primer A using 25 ng and 50 ng of DNA; Primer B using 50 ng of DNA), three of the four plant samples showed Ct values found in the same order that were seen with Primer B at 25 ng. For example, in each of those three runs, P. pratensis always had the lowest Ct value, while Z. mays had the highest one. In addition, the C3 grass (P. pratensis) had lower Ct values than the C4 grass (D. sanguinalis) in three of the four PCR runs. Finally, in all four of their runs, the non-grass C3 plant (A. saccharum) had a lower Ct value than the non-grass C4 plant (Z. mays). The 25 ng products indicate that both C4 plants had higher rbcl concentrations than C3 plants. Both blanks had considerably high Ct values, indicating very low concentrations of double-stranded DNA. In brief, a broad observation of the data, shows no immediate trends, but as Figure 5 shows, some trends are visible that support our hypothesis that C3 plants had lower Ct values and thus higher concentrations of the rbcl gene than the C4 plants.
Using protein standards a calibration curve was created to find the protein concentrations of equal masses of C3 and C4 plant samples. The average concentrations of C3 plants (19.856 and 49.584 mg/ml for A. saccharum and P. pratensis respectively) were in fact greater than those of the C4 plants (9.039 and 22.655 mg/ml for D. sanguinalis and Z. mays respectively). The 150 ug of our samples that were loaded onto our gel should have been more than enough for them to be identified as bands when run on a gel or Western Blot. However, the presence of our 50 kDa rubisco large subunit could not be verified using neither Comassie Blue gel electrophoresis nor a Western Blot. Therefore, one of the following two scenarios were likely to have occurred: 1) There was never much protein extracted from our samples and much of the absorbance was due to a pigment absorbing light at the same wavelength as our proteins or 2) although the absorbance measured during the extraction was accurate, the proteins were significantly degraded by proteases by the time that we loaded and ran our gels afterward. Either way, the results from our protein quantitation and identification scheme were inconclusive.
In conclusion, data from our DNA experiments strongly supported our hypothesis that there would be greater concentrations of rbcl gene in our C3 plant samples than in our C4 plant samples. However, the high absorbances obtained from our protein samples were not supported by the vacant gels or Western Blots that were run. In future experiments, more care would be taken to find a wavelength where only our proteins and no other pigments absorbed light, or to purify our proteins even more to get rid of molecules that would interfere with our true protein absorbances. Care would also need to be taken to make sure that if proteases were responsible for our spotless gels, we would need to either add inhibitors that would not degrade our gels or make sure that futher purification procedures were implemented to remove the proteases. Furthermore, in order to confirm even our positive results, more trials would need to be run with subsequent statistical analysis to verify that our data was not just accurate, but consistent. Future experimentation that tested rubisco and rbcl gene levels in different parts of C3 and C4 plants would also allow us to strengthen our hypothesis that C3 plants have higher concentrations of rubisco and rbcl.