The study of amino acid biosynthesis in microorganisms has profoundly affected our understanding of genetic regulatory mechanisms. However, very little is known about the way multicellular organisms, including animals and higher plants, regulate amino acid biosynthesis. This proposal describes genetic and molecular biological approaches to the study of tryptophan biosynthesis in the plant Arabidopsis thaliana. Tryptophan is required for synthesis of animal neurotransmitters and plant hormones (auxins). The characterization of tryptophan mutants and biosynthetic genes will permit the elucidation of the steps in the tryptophan and auxin biosynthetic pathways and provide a genetic basis for the study of hormone action during plant development. An important result of these experiments will be the development of molecular genetic system to study the regulation of gene expression in a multicellular organism. The organization and regulation of mRNa levels of the duplicated ARabidopsis tryptophan synthase B genes will be investigated by a number of complementary methods. The DNA sequence of the genes, and a mutation that appears to affect tryptophan synthase B activity will be determined. The expression of these genes will be assayed by RNa blot hybridization, primer extension and nuclease protection. The subcellular localization of each protein will be tested by two methods: uptake of trp synthase B in vitro translation products into isolated chloroplasts and immunolocytochemical localization. Since the trp2-1 mutant, presumed defective in tryptophan synthase B activity, is a conditional auxotroph, this mutant will be remutagenized to isolate secondary mutations that render the plant unconditionally auxotrophic. It is anticipated that this approach will identify mutations in both of the duplicated trp synthase B genes. The anthranilate PR-transferase structural gene will be cloned by heterologous probing of an Arabidopsis DNA library. This gene will be used in promoter mutagenesis studies with the trp1-1 mutant to define its cis-acting regulatory elements. MUtations that alter the expression of these, and other tryptophan biosynthetic genes will be identified by selection for resistance to 5-methylanthranilate and screening for anthranilate-accumulating mutants (by identification of blue fluorescent plants). A detailed molecular analysis of these mutants will reveal the transacting factors involved in regulating amino acid biosynthesis. A screen for anthranilate synthase mutants will be made by identification of nonfluorescent revertants of the blue fluorescent trp1 mutant.
