Introduction
The most abundant known protein is ribulose bisphosphate carboxylase (Rubisco). Rubisco is the most prevalent protein found in leaves, where it captures and fixes carbon via the photosynthetic pathway. Rubisco is a complex protein consisting of 16 total subunits. The Rubisco large subunit (RBCL) is one of eight large subunits encoded by the chloroplast genome. Chloroplasts are one of three types of plastids (the other two being chromoplasts and leucoplasts). Proplastids are plastid precursors that are capable of developing into any of the three plastid types.
Prior Rubisco research shows that Rubisco concentration and activity in Petunia hybrida corollas (flower petals) were approximately 30% of those seen in green leaves.1 Thus it is expected that translation of the RBCL gene would also be greatest in green leaves when compared to flowers. Prior RBCL research also shows that RBCL protein and mRNA levels in Beta vulgaris (sugar beets) are unaffected by drought conditions.2 In addition, prior research demonstrates that tobacco leaves utilize the catalytic inhibitor 2-carboxyarabinitol-1-phosphate (CA1P) to halt photosynthesis both at night and during drought conditions.3 Thus it is expected that the level of transcription of the RBCL gene during drought conditions would be similar to the level of transcription seen during normal conditions; however, it is expected that expression of the Rubisco protein (including RBCL) would be decreased during drought conditions.
The goal of the proposed study is to observe where RBCL expression occurs in chrysanthemums as well as how RBCL expression changes in chrysanthemums in response to drought conditions. This will be accomplished by depriving one of two chrysanthemum plants of water for five days (one plant will be watered for five days following purchase from Wal-Mart while one plant will not be watered at all for five days) after which the leaves and flowers will be analyzed for RBCL expression using Real-Time PCR and Western Blots.
Two specific hypotheses are proposed in this study. One hypothesis is that RBCL is expressed primarily in the leaves of chrysanthemums. It is predicted that transcription and translation of the RBCL gene will only be detected in leaves rather than flowers. In order to test this prediction, DNA will be isolated from the chrysanthemums followed by Real-time PCR (to quantitate how much DNA is present) and gel electophoresis (to visualize the PCR product). Real-time PCR will be used to detect the amplification of the RBCL gene. Isolated protein from the chrysanthemums and perform a Western Blot to quantitate how much RBCL protein is found in the leaves versus the flowers.
The second hypothesis is that the amount of moisture present affects RBCL activity, and more available water will result in more RBCL. It is predicted that the level of transcription of the RBCL gene will be similar in both types of chrysanthemums (those exposed to normal moisture conditions and those exposed to drought conditions) while the level of translation of the RBCL subunit will be lower in chrysanthemums exposed to drought conditions. Real-time PCR and Western blotting techniques will again be utilized to quantitate how much DNA and RBCL protein (respectively) is found in the watered chrysanthemums versus the chrysanthemums exposed to drought conditions.