Gene Networks and Chromatin Regulation of Phaseolin Transcription

Texas A&M Research Foundation, College Station TX

Investigators

Abstract

Intellectual merit Seed storage proteins are vital sources of food for mankind and livestock. Phaseolin is the most abundant protein stored in the seeds of the French bean, Phaseolus vulgaris. Genetically, it is encoded by a small family of similar but not identical genes, one of which (phas) has been studied in great detail. This NSF-funded research has the long-term goal of gaining an understanding of how specific genes are activated at certain times in defined tissues, and has led to major advances in plant molecular biology. These include the isolation and in vitro translation of plant mRNA, the first demonstration of introns in a plant gene, and the first functional transfer of a developmentally regulated gene (phas) from one species to another (bean to tobacco). Transcriptional regulation of phas is constrained both spatially and temporally. It was found that the establishment of a repressive chromatin structure and a rotationally positioned nucleosome over three phased TATA boxes of the phas promoter is responsible for the lack of phas expression in vegetative tissue. However, using an estradiol-inducible system for PvALF production and externally supplied abscisic acid (ABA), it was possible to induce activation of phas promoter-driven GUS reporter expression in leaves of Arabidopsis plantlets homozygous for two transgenes (pER8/XVEHA.PvALF). These experiments revealed that the activation of phas is a two-step process that involves nucleosome remodeling initiated by the seed-specific B3 domain transcriptional activator, PvALF, followed by abscisic acid (ABA)-driven activation of robust GUS transcription from the phas promoter that is easily identified by blue-colored leaves upon histochemical staining. Specific objective 1 of this project is to identify proteins that interact with the PvALF effector to achieve chromatin remodeling. In addition to yeast two-hybrid screening and immunoprecipitation with antibody against HA-tag, a novel approach will be to extract RNA from Arabidopsis leaves at specific time points during the potentiation and activation steps. High throughput sequencing of cDNA reverse-transcribed from the RNA samples will allow identification of the genes that are turned on in these steps, and the order in which they appear. In objective 2, further dissection of the various events governing the potentiation and activation of the phas promoter will be undertaken. Specifically, it will be determined if histone H3 depletion or substitution is intrinsic to effector-mediated activation of phas expression. The key role of PvALF and related effectors in initiating transcription from seed-specific promoters makes it imperative to search for the critical event that induces transcription from PvALF and its relatives (objective 3). Broader impacts In addition to gaining new information on molecular processes of gene expression, this project has relevance to understanding how the production of the seed protein of an important food crop is regulated. This project will provide training for a postdoctoral research associate, a graduate student and will give undergraduate students opportunities for laboratory experience. Several aspects of the proposed studies (e.g. cDNA cloning, yeast one/ two hybrid screening and T-DNA mutagenesis experiments) are expected to provide exciting opportunities for undergraduate participation. Arabidopsis transgenic lines or mutants created from the proposed studies (e.g. transgenic line expressing Myc-tagged histone H3 or H3.3) will also be made available for researchers in the plant community. Students of different heritage and gender will be encouraged to participate.