The long term goal of the proposed research is to understand the molecular basis of development in higher plants. The approach is to determine how a developmentally regulated gene is activated at specific times and in specific tissues. The gene for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is an essential plant gene with a well characterized promoter region and a bimodal tissue-specific expression pattern. EPSPS RNA is expressed at high levels in mature flowers with a dramatic increase in RNA abundance as the flower opens. A cDNA clone for a factor that binds specifically to the EPSPS enhancer region has been isolated. The cloned factor has the characteristics expected of the principal regulatory factor of EPSPS expression in mature plants-its RNA has a tissue-specific expression pattern that is very similar to that of the EPSPS gene. Because EPSPS is expressed in specific tissues in seedlings as well as in flowers, EPSPS serves as an interesting model system to study combinatorial control of gene expression in plants. Cis-element analysis in transgenic plants suggests that combinations of cis-elements that bind homologous or heterologous trans-factors may play a central role in regulating EPSPS expression. Characterization of EPSPS regulation using a newly developed in vitro transcription system from plants combined with in vivo characterization should provide insight into the combinatorial code that controls gene expression throughout plant development.
The specific aims of the research are to: 1) identify the DNA sequences responsible for developmentally regulated expression through use of reporter gene expression in transgenic plants and in transient assays with microprojectiles; 2) characterize the binding specificity of a cloned factor, EPF1 by determining the binding specificity of two widely separated zinc finger motifs in EPF1; 3) characterize the transcriptional activating ability of EPF1 with a plant in vitro transcription system; 4) determine the nature of the regulation of the EPF1 gene by analyzing the genomic clone of the factor for the determinants of tissue specific expression; 5) characterize the factors responsible for expression in seedling roots by analyzing nuclear extracts to identify factors that interact with the cis- elements controlling root cortex expression; 6) localize and determine the abundance of EPF1 protein in tissues by generating antibodies to EPF1 to define the abundance and localization of EPF1 protein; 7) characterize the transcriptional activating ability of EPF1 in vivo through introduction of overexpression and anti-sense constructs of the cloned factor coding sequences into transgenic plants to analyze the effect on EPSPS gene expression and to determine whether modulation of factor concentration leads to phenotypic changes; 8) identify the genes whose products interact with the factor by employing a genetic screen for suppressors of phenotypic alterations produced by abnormal factor expression to identify genes that interact with the factor or are activated by the factor.
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