Dr. Last proposes a 5-year study to continue his work on elucidating the mechanisms of amino acid biosynthetic regulation in Arabidopsis thaliana. Tryptophan biosynthesis has been chosen as a model system because it is highly amenable to genetic studies in Arabidopsis and because it has been well-characterized pathway in both prokaryotes and lower eukaryotes (yeast, Neurospora). Only within the last six years has significant progress been made in understanding this process in higher plants. Mainly through the efforts of the investigator's laboratory, most of the biosynthetic genes and corresponding mutant loci have been identified and characterized in Arabidopsis. Mutant lines, gene probes, and immunological agents are now available to undertake a detailed characterization of the regulation of tryptophan biosynthesis. The current proposal is focused more on the regulation of the pathway rather than on the function of biosynthetic enzymes. A number of details concerning the tryptophan biosynthetic pathway in Arabidopsis remain to be elucidated. For instance, the last enzyme involved in the conversion of anthranilate to tryptophan, indole-3- glycerol phosphate synthase (IGPS), has not yet been fully characterized. Arabidopsis cDNA clones which suppress a mutation in an IGPS-E. coli strain have been isolated; sequencing, Southern and Northern analysis, and genetic mapping are ongoing. If no mutations in the IGPS gene(s) are identified, an antisense construct will be used to investigate the function of IGPS-like cDNAs. The regulation of tryptophan synthesis will be investigated in several ways. The normal regulation of synthesis of pathway enzymes and adaptive responses will be examined in wildtype and structural gene mutant backgrounds in response to perturbation of amino acid levels, a strategy used successfully to study regulation of amino acid biosynthesis in yeast. Amino acid starvation, growth on inhibitors of amino acid biosynthesis (at levels which reduce the growth rate), culturing leaky amino acid mutants on minimal media, and growth on unbalanced amino acid mixtures will be utilized to perturb amino acid levels; mRNA and protein levels of tryptophan biosynthetic enzymes will be measured to ascertain the normal pattern of regulation and alterations in the responses of mutants. Regulatory mutants will also be identified by the screening for simultaneous resistance to 5-fluoroindole (resulting from reduced tryptophan synthase activity) and loss of blue fluorescence (reduction in the activity of enzymes preceding PAT in the pathway). In plants which fulfill both phenotypic criteria, the mRNA and protein levels of tryptophan biosynthetic enzymes will be investigated and the lesions genetically characterized. Monogenic non-trp allelic mutations will be further investigated. The last section of the proposal deals with the cell-type and subcellular localization of tryptophan biosynthetic enzymes which will be examined by immunocytochemistry. Functional assays will be used to investigate cell-type specific tryptophan biosynthesis. Genes encoding tryptophan biosynthetic enzymes will be fused to promoters with known patterns of expression and introduced into trp mutants defective in that enzyme. Suppression of the mutant phenotype will indicate that specific subsets of cell-type expression are sufficient for biological activity. Transport of tryptophan and its precursors will be investigated by genetic mosaic analysis designed to determine whether the blue fluorescence in trp1 mutants is cell autonomous. These mosaics will be created by gamma irradiation of seeds heterozygous for trp1 and a linked chlorophyll deficiency mutation (chlX).
