New Page 1

In this experiment, we investigated the relationship between petal pigmentation and RUBISCO content in chrysanthemum. RUBISCO is an enzyme found in plant chloroplasts, which initiates carbon fixation in the Calvin cycle. Previous studies have demonstrated increased levels of carotenoid and decreased levels of chlorophyll in pigmented tissue of plants. We believe that this relationship is due to the differentiation of plant plastids from the chlorophyll producing chloroplasts to the carotenoid (pigment) producing chromoplasts. We hypothesized that RUBISCO protein content would be highest in high chloroplast containing tissue samples (i.e. leaves) and would decrease in the high carotenoid/low chloroplast pigmented tissue samples. We also hypothesized that the color intensity correlated with carotenoid content- the more vibrant purple petals would have the highest carotenoid content, followed by lower concentrations in the orange petals and the white petals. Therefore, RUBISCO protein content would be greater in the white petals, followed by orange and purple. We expected that RUBISCO DNA would be relatively preserved across the tissue samples as chloroplasts and chromoplasts possess the same plastid genome.

Results from the real-time PCR using the 25 ng samples supported our contention that RUBISCO DNA is relatively preserved across the tissue samples as all of the tissue (leaf and petal) samples had similar CT values, suggesting similar DNA content (Table 4). The results of the 50 ng samples did not corroborate the 25 ng results as only the leaf samples were amplified (Table 5). Additionally, the CT values of the 50 ng leaf samples were similar to the CT values of the 25 ng leaf samples. We expected a lower CT value from the 50 ng samples, because they putatively contained twice the DNA content of the 25ng samples and should have amplified much sooner. Interestingly, two of the three 50 ng leaf samples produced near identical CT values as their 25 ng counterparts (data not shown). We suspect that the 25 ng data is more accurate than the 50 ng data, because the DNA was meant to be diluted prior to PCR. EDTA is present in the TE buffer which is used to re-suspend the DNA after isolation. EDTA also binds up the Mg²⁺ which is essential for PCR amplification. Because the DNA concentration levels were so low after isolation (53-119 ng/uL), further dilution could not be performed. Therefore, twice the volume of EDTA was present in the 50ng sample compared with the 25 ng sample and these elevated levels likely disrupted the amplification process more in the 50 ng sample.

We were not able to support or disprove our hypothesis regarding pigment and RUBISCO contents based on SDS-PAGE and Western blotting. Comassie blue staining of one SDS-PAGE gel did not reveal any bands. Degradation of protein by proteases may be responsible for the lack of bands found on the gel. Defective protease inhibitors could also be an attributing factor to the absence of bands. The Western blot did reveal three faint bands associated only with the leave samples (Figure 2). The faintness of the bands and the lack of bands for petal samples may be indicative of some protein degradation in the samples. Absolute confirmation that the bands are indeed RUBISCO protein could not be made because the RUBISCO standard did not reveal immunoreactive luminescence (Figure 2). However, since the antibody used specifically binds to RUBISCO, the bands shown on the Western Blot are likely due to the presence of this protein.

Studies done on plastid conversion from chloroplasts to chromoplasts have found that DNA copy number remains relatively constant during conversion. (Marano, 1992) With close Ct values across all tissue types we were able to demonstate that RUBISCO DNA content is relatively similiar in plant tissues regardless of pigment. RUBICO concentration positively correlates with chloroplast number, so although the data on protein content was unsupportive, the leaf tissue likely has higher RUBISCO protein expression, due to the importance of the chloroplasts in plant leaves for carbon fixation. (Hew et al, 1998).

Further investigation into the relationship between pigmentation and RUBISCO content in Chrysanthemums would be advantageous in order to gain a firm understanding of the effects of carotenoid levels on chloroplast content. Repeating our experimental procedures on a variety of pigmented plants could be useful in testing our original hypothesis. By using a wide-variety of flowering plants, it would allow us to observe a range of pigments. This would also permit us to observe pigments in a number of different plant species, instead of one species. Relationships between different species of plants, and their pigment and chloroplast content, may prove to be more useful in distinguishing a connection between carotenoid level and RUBISCO expression.