Dissection of cis-regulatory Regions in Plants Using Recombination, Allelic Expression, and Site-directed Mutagenesis

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Intellectual Merit. Central to the understanding of organismal development are the underlying genetic mechanisms that drive variation in gene expression. It is generally agreed that sequence variation in the regulatory regions of genes plays a major role in expression divergence and organismal diversity, yet little is know regarding the structure and function of these complex promoter regions. This project takes a step toward resolving the genesis of this expression variation by dissecting the functional modules of regulatory regions into their controlling units. Evidence from a wide range of eukaryotic organisms indicates that, within individual species, variation in allelic expression is a common phenomenon and occurs at a significantly high frequency in various tissues and during different developmental stages. Using two resource-rich model plant taxa, Arabidopsis thaliana and Zea mays (maize), variation in allelic expression, combined with naturally occurring meiotic recombination, will be used to map the regulatory components of these expression differences for several genes in various plant tissues. The data are expected to reveal the regulatory components of allelic expression variation in two distinct plant lineages and contribute significantly to a more complete understanding of the structure and function of plant promoters. Additionally, other regulatory components of gene expression that do not contribute to allelic expression differences, but do function as cis-regulatory controls, are expected to be discovered using in situ site-directed mutagenesis in upstream regions of the same genes in Arabidopsis. These data, combined with those from the allelic expression portion of the project, will provide a detailed characterization of plant promoter structure and function in the Arabidopsis and maize regions of interest. The completion of this project will contribute to a broader understanding of promoter evolution in plants, specifically with respect to the role of module swapping via meitoic recombination, and will also provide a promising methodology for elucidating mechanisms for the cis-regulatory control of plant gene expression. This project promises not only intellectual advancement in the fields of molecular and evolutionary genetics, but also the development of a number of possible practical industrial technologies designed to enhance and advance agricultural practices.Broader Impacts. This research lends itself to multiple activities involving outreach, the inclusion of members of underrepresented groups, and scientific training of students at all levels. In the state of West Virginia, a small fraction of high school students attend college (~16%), and 20% of those incoming undergraduates are first-generation college students. These projects will provide multiple educational opportunities for these underrepresented students via scientific training in laboratory practices that lead to landmark discoveries, and ultimately to the implementation of innovative real world agricultural applications that benefit society as a whole. Other underrepresented groups will be provided opportunities at WVU via the McNair Scholarship, designed to provide support for students from disadvantaged backgrounds, and the NSF Research Education for Undergraduates (REU) program. Also, a number of educational outreach opportunities exist at WVU via the program for Women in Science and Engineering (WiSE), and the Summer Undergraduate Research Experience (SURE) program. Students will be provided the opportunity to disseminate the results of their work among the broader scientific community at annual international meetings. In addition, this research is designed as part of the WVU Computational Biology Initiative, an interdepartmental effort among the Departments of Biology, Mathematics and Statistics to create undergraduate and graduate programs in the emerging field of bioinformatics. All scripts and data generated will be used to develop new courses under the Initiative.