| In plants, the tryptophan biosynthetic pathway leads to the synthesis of the amino acid tryptophan and a number of important secondary metabolites, including the growth regulator indole-3-acetic acid (IAA) and various defense compounds. These metabolites differ in spatial distribution, abundance, environmental responsiveness, and developmental control. To control the accumulation of these diverse compounds, plants have evolved elaborate mechanisms of regulating tryptophan primary and secondary metabolic pathways.; To understand the molecular mechanisms involved in tryptophan pathway control, we conducted a genetic screen for a&barbelow;ltered t&barbelow;ryptophan r&barbelow;egulation mutants. A recessive atr4 mutation was positionally cloned and found to be a loss-of-function allele in a cytochrome P450 gene, CYP83B1, which encodes an enzyme involved in tryptophan secondary metabolism. The atr4 mutant was characterized using a combination of biochemical, molecular, and genetic approaches. Gene expression profile analysis revealed elevated levels of ATR1, a Myb transcription factor, in the atr4 mutant background. An overexpression allele of ATR1, atr1D, had been previously identified through the atr genetic screen. Therefore, the activated tryptophan pathway phenotypes of atr4 are most likely mediated through ATR1. To further understand factors important for tryptophan pathway activation in atr4 mutants, we conducted a genetic suppressor screen for 5-methyl t&barbelow;ryptophan r&barbelow;esistance s&barbelow;uppressor (trs) mutants. Isolation of five trs complementation groups suggests that there may be many possible mechanisms leading to the suppression phenotypes observed in the trs atr4 seedlings.; Besides atr4 and the atr1D Myb overexpression mutant, the atr screen yielded the atr2D mutant. The atr2D mutation is an altered function allele of a basic helix-loop-helix (bHLH) transcription factor. Given a strong precedent for Myb factors to work together with bHLH proteins in plants, we tested for interactions between ATR1 and atr2D. A combination of molecular and genetic approaches was used to determine that the atr1D and atr2D mutations activate the tryptophan pathway through independent mechanisms.; The results presented in this thesis further our understanding of tryptophan biosynthetic pathway regulation in Arabidopsis thaliana. Dissection of the regulatory network perturbed in the recessive atr4 mutants has elucidated fundamental plant strategies for metabolism adjustments in response to environmental cues. The dominant atr mutants, atr1D and atr2D, provide insights into two distinct pathways that can be modulated to confer coordinate activation of multiple tryptophan biosynthetic genes. Therefore, the dominant atr mutants may find future application in metabolic engineering efforts to enhance the nutritional value of agriculturally important crops. |
