Introduction
The protein ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known as “Rubisco,” is the most abundant enzyme found in plant leaves. It is also believed to be the most abundant enzyme on the earth. Rubisco plays an integral role in the fixation of carbon, as it is utilized in the Calvin cycle to catalyze the reaction between carbon dioxide and the ribulose bisphosphate molecule, containing five carbon atoms. The resulting molecule contains six carbon atoms, and then splits into two phosphoglycerate molecules, containing three carbon atoms each. The enzyme catalyzes the first major step of the process of carbon fixation. As the Rubisco enzyme can fix either atmospheric oxygen or carbon dioxide and is nonspecific, there are two major pathways in its functioning. First, the enzyme can catalyze the fixation of carbon dioxide using carboxylase to produce phosphoglycerate. Second, the enzyme can use oxygenase to catalyze the fixation of oxygen, and produce phosphoglycerate along with glycollate-2-phosphate.
Rubisco is made up of eight identical subunits comprising each of two chains: one large, made of eight large subunits, and one small, made of eight small subunits. The large polypeptide chain is known as “L,” weighs about 55,000 daltons (55 kd), and is encoded by the rbcL gene found in chloroplasts. The small polypeptide chain is known as “S,” weighs about 13,000 daltons (13 kd), and is encoded by the rbcS gene found in the nucleus.
Many studies have been done relating to levels of expression of rubisco at varying times during the day in oak leaves (Jorge et al, 2005) and in different tissues of a plant such as a tomato. This is caused by differential regulation by a promoter subfamily for the rubisco small subunit genes in tomato (Meier et al, 1995).
Our experiment involved leaf samples from two types of trees: coniferous and deciduous. Leaves of red oak, Quercus rubra, and maple, Acer saccharum were selected and used as specimens for deciduous trees. Holly, Ilex opaca was selected as a specimen for the category of coniferous trees. Our hypothesis was that the needles of a coniferous tree would have a greater amount of Rubisco enzyme than the leaves of a deciduous tree because they do not become dormant during winter. The coniferous tree would have a greater amount of rubisco enzyme being expressed, as it would need to produce the enzyme to fix carbon year round as opposed to a cessation of carbon fixation along with a lack of rubisco expression during the winter months in a deciduous tree.
Several different techniques were used to test our hypothesis, including methods to analyze both DNA and protein. Techniques utilized to analyze DNA included real-time polymerase chain reaction (PCR) and an agarose gel, and techniques used to analyze protein included a western blot analysis, an electroblot technique, and an acrylamide gel. Optical density was measured using spectroscopy to quantify both DNA and protein samples.
REFERENCES
Jorge, I, Navarro, R., Lenz, C., Ariza, D. The holm oak leaf proteome: analytical and biological variability in the protein expression level assessed by 2-DE and protein identification tandem mass spectrometry de novo sequencing and sequence similarity searching. Proteomics. 2005 Jan;5(1):222-34.
Kellogg, Elizabeth and Juliano, Nickolas. 1997. The structure and function of Rubisco and their implications for systematic studies. American Journal of Botany, 84:413.
Meier, I., Callan., K., Fleming., A., Gruissem., W. Organ-specific differential regulation of a promoter subfamily for the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit genes in tomato. Plant Physiology. 1995 Apr;107(4):1105-18.
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