Amino acids are essential molecules in plant metabolism. Amino acids carry reduced nitrogen while serving as precursors for protein synthesis and secondary metabolites. Translocation of amino acids in the cell is mediated by amino acid transporters. While about 100 transporters have been identified, only a dozen have been fully characterized. The regulation of amino acid transporters is not fully understood and stands as the basis of this study. Previous toxicity-based screenings of Arabidopsis thaliana mutants led to the isolation of a loss-of-function line and the phenylalanine insensitive growth (pig1) mutant capable of growth on toxic concentrations of phenylalanine (1). The pig1-1 mutants also displayed a deregulated metabolism (1). We followed this work with a similar forward genetic screening of Arabidopsis thaliana that led to the identification of 18 mutants capable of growth in the presence of amino acids at toxic concentrations. From this screen, seven mutations were confirmed to affect the amino acid transporter AAP1. Here I demonstrate that, when expressed in yeast deficient for endogenous amino acid transporters, three variant aap1 proteins restored growth similar to yeast complemented by wild type AAP1. Transport of radiolabeled Pro was abolished by variant aap1 proteins while deletion of an intracellular loop spanning the 8th and 9th transmembrane domains reduced Pro transport in yeast. Site directed mutagenesis of this loop conferred a variant aap1 protein which augmented Pro transport in yeast. Amino acid transport in loss-of-function aap1 plants display decreased uptake and increased efflux. In addition, aap1 mutant plants accumulated between 2 and 8 times more free amino acids in the leaves than the wild type. These observations are not fully compatible with the accepted role of AAP1 in transport by the root. The present work describes how the amino acid transporter AAP1 could play a role in regulating amino acid metabolism. We hypothesize that the amino acid transporter AAP1 functions as a senor that is involved in amino acid homeostasis in addition to its established role as a transporter. Is true, this would make AAP1 the first identified amino acid sensor in plants. Knowledge of the mechanism of amino acid sensing would enable us to engineer crops for improved nutrition in a more efficient way than affecting metabolic enzymes. / MSLFS / Amino acids play essential role in crop metabolism. Amino acids are nitrogen containing molecules that are used to make protein and many other molecules. They are located through-out the plant and move from organ to organ by amino acid transporters. A dozen of approximately 100 known amino acid transporters have been studied in depth and are well understood. Interestingly, not much is known about these transporters and what controls their activity. A mutant weed, Arabidopsis thaliana mutant phenylalanine insensitive growth (pig1), was identified by its ability to survive in toxic environments with high amounts of the amino acid phenylalanine and also showed an irregular metabolism of amino acids (1). The Pilot Lab and I were able to identify 18 more mutants with similar abilities to survive in toxic amino acid conditions by performing similar experiments. Seven of the new mutants were found to have mutations that effected the amino acid transporters AAP1. Using yeast incapable of growing in nitrogen restricted conditions where amino acids are the only source of nitrogen, I found that three of the variants app1 proteins we identified were able to restore growth like wild type AAP1 yeast. These variant aap1 yeast did not show the ability to transport the acid proline, while other alter versions of the aap1 protein made to alter its structure and proposed significant parts were able to increase proline transport. Plants with no or mutant aap1 proteins showed a decreased ability to uptake amino acids in addition to increased efflux of amino acids. These plants also had a higher level of amino acids in their leaves than normal wild type plants. These results obtained in both plants and yeast with altered amino acid transporter aap1 do not agree with what we understand to be the accepted function of AAP1 transporting amino acids in plant roots. The work presented in this thesis discusses how AAP1 could be involved with controlling plant amino acid metabolism. It is my hypothesis that the transporter is serving two functions by both transporting and sensing amino acids. As a sensor, AAP1 serves to maintain a proper balance of amino acids for plant metabolism. If AAP1 does this, it would be the first of its kind to be identified in plants and help enhance crop engineering for better nutrition to better feed growing populations.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/99368 |
| Date | 22 January 2018 |
| Creators | Boyd, Shelton Roosevelt |
| Contributors | Plant Pathology, Physiology and Weed Science, Pilot, Guillaume, Grene, Ruth, Bevan, David R., Sobrado, Pablo |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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