Master of Science
Kohalmi, Susanne E.
In Arabidopsis thaliana, a family of six AROGENATE DEHYDRATASE (ADT) enzymes catalyze the final step of Phe biosynthesis. While all AtADTs localize to chloroplasts, my work focuses on AtADT5, which is the only isoform also found in nuclei. Based on in silico evidence, I hypothesized that phosphorylation regulates AtADT5’s subcellular localization and function. Using bimolecular fluorescence complementation, I found that AtADT5 is a phosphoprotein in vivo. Further, I designed an experimental approach that will allow profiling all post‑translational modifications in AtADT5 using mass spectrometry. Lastly, I tested the effect of phosphorylation on AtADT5’s subcellular localization using phosphomimetics and found no change relative to wildtype. This work is the first demonstration that AtADT5 is phosphorylated in vivo and contributes to our understanding of the regulation of the ADT gene family.
Summary for Lay Audience
Phenylalanine is a building block of life, used by all organisms to make proteins. While humans must take up phenylalanine in our diet, plants can synthesize it themselves, using an enzyme called arogenate dehydratase (ADT). Phenylalanine is important in plants, not only to make proteins, but for the production of specialized metabolites, which are chemicals that help the plant survive in various ways, from helping respond to environmental changes to building molecules that support the plant structurally. In the plant Arabidopsis thaliana, there are six versions of AtADTs. All six of the AtADTs perform their role in the chloroplast; however, one version, AtADT5, is also found in nuclei. Currently, the role of AtADT5 in the nucleus and how it is being transported there are unknown. After proteins are synthesized, their amino acids can undergo a small chemical change, called phosphorylation, that can affect their stability and function. As AtADT5 was highly predicted to be phosphorylated through computational analyses, I hypothesized that phosphorylation is involved in transporting AtADT5 to the nucleus. The results of this thesis demonstrate that AtADT5 is phosphorylated, but that the phosphorylation status of the tested amino acids is not sufficient to alter AtADT5 nuclear localization. This work contributes to our understanding of the regulation of the ADT gene family and thus phenylalanine biosynthesis.
Barac, Eileen, "Evaluating Phosphorylation as a Potential Regulator of ADT5 in Arabidopsis thaliana" (2023). Electronic Thesis and Dissertation Repository. 9707.
Available for download on Saturday, October 25, 2025