Discussion

We hypothesized that older plants would have less Rubisco expression than younger plants. This was based on the action of senescing. As plants age and begin seed production energy is devoted to perpetuating the species. Thus, Rubisco expression falls as it becomes secondary. Arabidopsis thaliana was chosen for study because it is an annual plant, the life cycle is relatively short, and there was a high availability through the biology department.

Leaves were harvested at random from four different sets of plants, termed trays one through four. Tray one held the oldest plants which were flowering and had obviously senesced leaves. Trays two and three held plants which were bolting, but only tray two had begun to flower. Tray four held young plants which were a month old and had developed into leafy stubs.

Real-time PCR was conducted to determine the level of gene expression by analyzing Rubisco DNA. Samples of 50ng, 100ng, and 150ng were analyzed using primer A. We selected primer A because it was tried and true for RT PCR. Ct values were compared for each tray of plants. The RT PCR data from our experiments became splotchy and made it difficult to identify trends. The Ct values in figure 2 are only present for trays one and three. This is most likely due bad technique and not because of a lack of Rubisco coding DNA in the leaves. Table 4 indicate that most of the DNA we extracted had a decent purity (A260/A280 was close to 1.8) except for that of tray 4, which had a large amount of impurities giving it a ration of .7375. The Ct values for tray three are 100ng at 10.973 and 150ng at 11.892. These are clearly less than those for tray one, 100ng at 12.181 and 150ng tray 14.062. The Ct values are most likely greater for 100ng samples because the 150ng samples were two concentrated and repressed the replication process. A lower Ct value corresponds to a larger concentration of DNA. These values indicate that older seed producing plants have less Rubisco coding DNA in their leaves than maturing plants. We must also note that our blank had a Ct value of 13.217 (red line), which is the second quickest Ct value in our data. This is obviously a result of poor technique when establishing our PCR samples.

Further exploration of our PCR data using an agrose gel is shown as a figure 3, stained with sybr green . We observed that all of the samples which had Ct values contained DNA. Most notably we can see that the blank, lane E8, contained Rubisco coding DNA. Obviously, Rubisco coding DNA found in the blank sample indicates that our results should be taken with a grain of salt.

We also considered the expression of Rubisco by measuring the concentration of its large subunit in plant leaves using several methods. Our Western Blot, shown in figure 5, indicates the concentration of Rubisco large subunits, which appear around 47,600Da. Trays one and two have a weak concentration of the large subunit. Trays three and four indicate a substantial concentration of protein. Thus, tray one (older plants) having more Rubisco coding DNA than tray four (young plants). Protein is only produced after transcription and translation. This gives two possibilities of why older plants contain more large Rubisco subunits. It could either be that older plants just have more Rubisco coding DNA and a higher expression or that young plants have a greater level of expression, but just have not processed the DNA into protein.

Our Western Blot transfer membrane is displayed in figure 6. The data displayed here is consistent with that of the Western blot. The photo taken with a BioRAD Chemilumenescent camera reveals protein in trays two through four. Tray one appears to have no large Rubisco subunits in it at all. Again, this shows that developing plants contain more Rubisco protein than seed producing, senescing plants.

Our results for DNA and Rubisco protein analysis seem to be contradictory to one another, but our original hypothesis may hold true. The fact that our blank sample in RT PCR contained Rubisco coding DNA combined with a low DNA purity for tray four leads note that these test may be unreliable. However, more Rubisco subunits found in older plants does not necessarily indicate that their expression for the protein is greater. As mentioned before DNA must be converted to protein and it must also be noted that the degradation of Rubisco is not factored into our results. Our PCR data indicates that younger plants do contain more Rubisco coding DNA. We propose that younger plants do have a greater Rubisco expression based on this and the reason we see more Rubisco subunits in older plants is due to them having previously produced protein.

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