Discussion: Home Introduction Methods Results Literature Cited
Total protein extraction as visualized through polyacrylamide gel electrophoresis and comassie blue staining indicated the presence of protein in all tissue samples (Figure 2). The intensity of the stained protein bands indicated that more protein was present in the leaf tissue than in the tomato fruit tissues. Upon method review it was determined that this increased protein concentration in the leaf tissue of the tomato plant was due to increased amount of leaf tissue protein loaded. Due to this misstep, tomato leaf results are not comparable to the results obtained for the various tomato fruit tissue samples. Because of this and other problems occurring during leaf DNA extraction (Table 2), the data for the tomato leaf sample will not be compared to the other samples in this report.
Western blotting of the total tomato fruit protein indicated that the amount of RBCL present in the green and orange tomato fruit is similar and slightly higher than the levels observed in the red tomato fruit (Figure 3). This does not fully support our hypothesis that the level of Rubisco expression would be greatest in the green tomato fruit tissues and lowest in the red tomato fruit tissue. These results indicate that Rubisco expression levels are similar in green and orange tomato fruit tissues and do not decrease until the tomato has entered the late phase of the ripening process, and begun to turn red. This may indicate that Rubisco expression halts suddenly near the terminal phase of ripening instead of gradually over the entire ripening process as was hypothesized. The total red tomato tissue protein as visualized through comassie blue staining (Figure 2), however, appears to be lower than in orange and green fruit tissues, and may indicate that less red tomato protein was loaded during the procedure.
Quantification of the copy number of Rubsico gene present in red, orange, and green tomato fruit tissues using RT-PCR indicated that the number of RBCL copies is highest in the red tomato fruit tissue and lowest in the green tomato fruit tissue (Figure 4A). These results do not support our hypothesis that the number of copies of the Rubisco gene would remain the same in the various tomato tissues during the ripening process. Instead these results show that the number of Rubisco gene copies is actually increasing during the ripening process. A comparison of the results of the RT-PCR reaction (Figure 4A) to the levels of Rubisco protein expression occurring within the 3 tissues (Figure 3) indicates that although the number of copies of the Rubisco gene is greatest in the red tomato fruit, the level of RBCL expression is the lowest. This would indicate that although chloroplasts differentiate into chromoplasts during the tomato ripening process (Boyer et al., 1986) additional chromoplasts must be synthesized to allow for enough of the red tomato skin pigment to be produced. The synthesis of additional chromoplasts and the resulting increase in the total plastid number in the red tomato fruit tissue is suspected to have led to greater copies of the Rubisco gene being present in the tissue. A comparison of the level of Rubisco expression in the orange and green tomato fruit tissues (figure 3) with their numbers of Rubisco gene copies (figure 4A) indicates that the higher number of Rubisco gene copies present within the orange tomato fruit tissue is likely due to the additional chromoplasts needed for pigment production since both tissues have the same level of Rubisco protein expression. This would indicate that the first step in the tomato plant ripening process is to synthesize additional chromoplasts for pigment production and to later differentiate chloroplasts into chromoplasts through gene regulation.
Analysis of the melting curve of the RT-PCR product of the 3 tomato tissues (figure 4B) indicates that 2 products were present within the RT-PCR reactions. This was not unexpected due to the formation of primer dimers in the PCR reaction, and are hypothesized to be the cause of the lower melting product. The hypothesized primer dimer product was completely melted at approximately 83°C and therefore RT-PCR data was collected at 84°C to avoid the primer dimer data corrupting our results. The larger product melting curve indicated that the larger products were approximately all the same size and had similar guanine/cytosine contents. This indicated that the RT-PCR product amplified from the red, orange, and green tomato tissue DNA extracts was likely the same product in all the tissues.
For additional verification, the RT-PCR products of these tissues were run on an agarose gel (figure 5). The major RT-PCR product appears to be ~200bp. This is the expected product size for the PCR primers used to amplify the section of the RBCL gene targeted. These results support the conclusion that our RT-PCR amplified the correct RBCL target.
In conclusion, Rubisco production appears to decrease at some time point between when the tomatoes are orange and when they are red in color (~ between 1-2 weeks into the ripening process). The level of Rubisco expression in the early stages of the tomato ripening process appears to remain constant although the number of plastids is increasing. It is suspected that the increased number of plastids is due to the synthesis of new chromoplasts for red pigment production. At a later time in the ripening process it is also concluded that Rubisco production decreases by means of gene regulation as chloroplasts differentiate into chromoplasts. This knowledge may be applied to indoor farming of tomatoes, allowing for artificial light levels to be decreased when there is abundance of Rubisco present in the plant, and the plant has an increased photosynthetic capacity, and for light levels to be increased, as the Rubsico levels, and photosynthetic capacity of the plant decrease. Or, vice versa depending on the results of future studies. This would allow for indoor farmers to efficiently use artificial lighting to save money on yearly electric costs.
During this experiment we had difficulties with our DNA extraction procedure. In the future a different extraction procedure could be used when working with tomato fruit tissues. Most notably, we had issues with the extraction of DNA from the tomato leaf tissue as well as orange tomato fruit (Table 2). We also saw RT-PCR amplification in our negative control (Figure 4) which indicates our RT-PCR reaction was likely contaminated. Repetition of the RT-PCR reaction resulted in no observable amplification in any of the tomato fruit samples (Data not shown) and lends support to the possibility that the observable amplification in the first RT-PCR reaction was due to contamination and not actually biologically relevant. There were also issues with the DNA purity of our samples, but the proteins associated with our DNA did not appear to affect the RT-PCR reaction (Table 2). In the future a repetition of the full experiment using a different DNA extraction procedure would be useful, in addition it would allow us to determine the influence of the negative control contamination on our results.