Genetic Study of the Relationship of Clp Protease to Plastid Metabolism & DNA Repair in Arabidopsis Thaliana
Plastids play a critical role in cellular function by manufacturing metabolic compounds and conducting photosynthesis. These processes are maintained through various mechanisms, including proteolysis in the plastid. Understanding how proteolysis enhances growth could improve efforts to maximize crop yield. Indeed, the chloroplast can produce valuable chemicals and vaccines due to its high capacity for protein synthesis and could be improved by an understanding of plastid proteases that maintain protein homeostasis. Proteases are fundamental to plant development by their regulation of proteolysis, or the breakdown of proteins into smaller polypeptides. Clp Protease, the most abundant, soluble protease complex in plant plastids, is of particular interest to the Van Wijk Lab. Recent genetic studies suggest that the Clp protease contributes to protein biogenesis, embryogenesis, and cellular metabolism. Previously the lab identified eight candidate substrates that bind uniquely to ClpS, which is a substrate selector for proteolysis. This summer, I studied two proteins suspected to regulate DNA organization and repair in Arabidopsis thaliana – UVR and pTAC17. I focused on the localization and spatiotemporal expression of these proteins as well as how they affect plant phenotype. To isolate their role in plant development, I compared knockout mutants grown with wild type plants with intentions to perform the complementation test by floral dipping. Immunoblot analyses determined the subcellular localization of UVR and pTAC17 proteins in chloroplast stroma and membrane; compared their expression between wild type and ClpS mutant; and analyzed their expression levels in 1 to 6 week old leaves as well as flower, stem, and silique samples. The results of this project could further the understanding of their functions in plastid DNA maintenance and its regulation through theClp protease, which is necessary to expand our knowledge of protease function, its relation to DNA integrity, and thus overall plant growth.
My fascination with plant science stems from an awe of their capabilities and a respect for those who discover their potential. With the expertise of my mentors, I was able to take part in this scientific exploration. Two months ago Western Blotting was a foreign concept to me, but now this procedure and many others like Colony PCR and Bacterial Transformation, come naturally. My mentors and lab members instilled a desire to discover and become a better researcher. Working with them provided an awareness of how successful research is conducted and the ability to think critically about a project. This internship gave me skills I will continue to improve upon in future labs and ingrained a new perspective for my undergraduate years. Now I not only visualize myself pursuing scientific advancement, but also have a growing desire to achieve it.