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.
In 1665, Englishman Robert Hooke used a primitive microscope to examine cork and other plant tissues. He noticed that they contained regular patterns that reminded him of the small rooms or "cells" in which monks slept in monasteries. However, 100 years passed before it was recognized that cells represent the basic unit of life and yet another 100 years before The Cell Theory encompassed the fact that all cells arise from existing cells. We now know that the creation of new cells depends on the expression of information that is stored in genes, long stretches of DNA. Modern science permits chemical definition or "sequencing" of genes and testing how they work, i.e. how the information they bear is "expressed", not only to provide building materials for the new cells but also how these materials are made in, or delivered to specific sites and at specific times. This project was undertaken to learn how genes encoding seed proteins function. The seed protein used as a model in these studies is called phaseolin and it is the most abundant protein present in seeds of the common bean, Phaseolus vulgaris. We anticipate that the new information generated by these studies will be used to increase crop yields (e.g. by making the phaseolin [phas] promoter stronger), to improve the nutritional quality (e.g. by modifying the coding sequence), and to enhance disease resistance. These attributes make the phaseolin system especially valuable for increasing both scientific knowledge concerning life processes and practical applications for improvement of a major crop plant. In work conducted with NSF funding, we have made substantial additions to our basic knowledge of the way in which the phaseolin gene is turned on and have identified the ways in which multiple genes are involved for efficient expression of seed storage proteins. In nature, synthesis of phaseolin is temporarily and spatially confined to stages of development known as embryogenesis and microsporogenesis, being stringently turned off during all vegetative stages of plant development. PvALF (Phaseolus vulgaris ABI3-like factor) B3-type transcription factor is vital for transcription from the phas promoter. In the presence of abscisic acid (ABA), PvALF, and several other B3-type proteins, activate the expression of the phas promoter. Phas activation requires the interaction and coordination of epigenetic (nucleosome positioning and histone modification) and genetic (transcription factor) events. Under natural conditions in developing seeds, events associated with potentiation and activation of the phas promoter are inseparable. In order to separate these two events, we developed estradiol-inducible heterologous PvALF/phas-gus system in Arabidopsis. Potentiation (chromatin remodeling) is achieved by inducing PvALF expression in transgenic Arabidopsis with estradiol for 4 h [4E], followed by activation of the phas promoter with ABA-induced signal transduction cascade for another 4 h [4E4A]. We utilize this system, in conjunction with the powerful RNA-Seq approach to identify the individual components of gene networks turned on by PvALF to activate phas expression. We identified over 1300 genes, spanning 11 functional categories, whose expression coincides with changes in the transcriptional status of the phas promoter. Overall, the 4E4A dataset shows that PvALF and ABA trigger many seed-specific genes in leaves. Results from the network analysis revealed that PvALF has regulatory edges with RINGLET2 (RLT2) and AINTEGUMENTA-LIKE 5 (AIL5), which in turn may directly or indirectly regulate phas expression. Furthermore, loss-of-function genetic assays, confirmed the requirement of RLT2 and AIL5 for the efficient activation of phas-GUS expression in transgenic Arabidopsis leaves. Noteworthy, down regulation of RLT2 and AIL5 affects seed gene expression in seeds. Based on the results of these experiments, a specific model for PvALF-dependent phas activation is shown in Image 1, in which PvALF interacts with RLT2 to promote the potentiated state of phas chromatin through its interaction with CHROMATIN-REMODELING PROTEIN 11 (CHR11) [Step 1]. The rotational and translational positioning of the repressive nucleosome over the phas promoter might be randomized by CHR11 to facilitate nucleosome spacing, thereby providing a platform for the step 2 of phas expression (transcriptional activation). During the ABA-dependent activation step, PvALF may bind with several cis-elements present in the AIL5 promoter, thus activating the expression of the phas promoter. Overall, the identification of these key factors involved in the phas activation provides important insight into the regulation of seed-specific genes.
