Introduction

Carbon is the most essential element in the lives of all organisms.  As a result, one of the most fundamental methods for classifying living things is an examination of the mechanism by which they obtain their carbon.  Unfortunately, in nature, carbon is only present in the form of highly oxidized compounds, like minerals and CO₂ gas.  Therefore, organisms like plants have developed methods of fixing these oxidized carbon sources into organically useful forms, like molecules rich in carbon-hydrogen bonds.  Rubisco, the protein of interest in this experiment, plays a principal role in the carbon fixation performed in every plant.  In the cells’ chloroplast, Rubisco binds the free CO₂, linking it to a short 5-carbon chain.  Rubisco then cleaves the chain into two smaller chains, known as phosphoglycerates, which are used for metabolism in various cellular pathways.  While the majority of these phosphoglycerates are recycled to make carbon compounds necessary for the Calvin cycle, one in every six are used to make sucrose, which provides energy for the plant.  Some of this sugar is stored for later use, in the form of starch.

Courtesy of http://www.biology.arizona.edu/biochemistry/problem_sets/photosynthesis_2/graphics/01t.gif

Rubisco is a popular protein used in experimental research for several reasons.  Rubisco is a relatively inefficient enzyme; a limitation plants compensate for by building the protein in excess.  It follows that Rubisco is the most abundant protein on earth.  As a result, it is fairly easy to isolate the protein from plants.  Also, the protein is highly conserved among different plant species, which allows the experimenter to use the same treatments (i.e. primer sets, antibody selection, experimental pH) when working with the isolated protein. 

http://kuchem.kyoto-u.ac.jp/kozo/home/Image/rubisco/1mer_150.gif

            In this experiment, Rubisco levels were monitored in green and yellow leaves from the Sugar Maple (Acer saccharum Marsh) and the Green Ash (Fraxinus pennsylvanica).  The leaves used were gathered from trees on the James Madison University campus.  As mentioned above, leaves produce sugar for the rest of the plant by using CO₂ and light photons, during the spring and summer months.  During autumn, the days become shorter and the nights become longer, a seasonal change that results in the plant receiving less light.  Among other things, this change results in a corky membrane that forms between the tree’s branches and stems, which cause a significant decrease in nutrient flow.  As nutrient levels decrease, the plant stops producing chlorophyll, a molecule that absorbs sunlight and gives the leaf its green color.  As a result, the leaf’s green color fades to a rich yellow, caused by carotene, a molecule that is always present in plants.  Chlorophyll degradation results in the plants sugar production to cease.  The leaf then lives off the starch it had been storing, until it eventually turns brown and dies. 

http://www.lesinsectesduquebec.com/plantes/acer_saccharum-1.JPG

    The hypothesis for this experiment was that the green plants would have higher Rubisco levels then yellow plants, in both leaf species tested ( Acer saccharum and Fraxinus pennsylvanic). This hypothesis was tested by isolation of protein and DNA, performing real time PCR analysis by agarose gel electrophoresis, and running a western blot.     

http://www.uwgb.edu/biodiversity/herbarium/trees/frapen_leaf01.jpg

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11/21/2005