Lucia C. Strader, Ph.D. - US grants

PROJECT SUMMARY - INTERACTION OF PHYTOHORMONE SIGNALING PATHWAYS Candidate: The candidate's long-term career goal is to become a principal investigator in an academic setting, studying the interaction networks involved in phytohormone response. During the training period, she will learn biochemical research approaches to complement her graduate applied agricultural and post- doctoral molecular genetic research. Training Environment: Rice University provides an ideal training environment because of its quality research and intimate setting. Dr. Bonnie Bartel, the mentor for this project, is a leader in Arabidopsis research. Further, she is an active mentor who devotes the majority of her time to her research and her postdocs and students. Research: The long-term goal of this project is to enhance understanding of the signaling network that connects the phytohormones auxin, abscisic acid (ABA), and ethylene. The dual-specificity protein phosphatase, IBR5, the subject of this proposal, is a novel point of interaction between auxin, ABA, and ethylene signal transduction networks. Several interconnected approaches will use IBR5 to gain greater understanding of the relationship among these phytohormones. Firstly, IBR5-interacting proteins will be identified (Aim 1); these may include substrates and regulators of the IBR5 phosphatase. Additionally, second-site ibr5 modifiers will be analyzed (Aim 2) to determine whether sensitivity to auxin, ABA, and ethylene can be separated, or if they are interconnected in inseparable ways. Identification of defective genes in these modifiers (Aim 3) will allow molecular elucidation of interactions between these phytohormone pathways. These studies will contribute to understanding of which signaling components are shared among these three pathways and which are not shared, and will identify additional nodes in the phytohormone signaling network. In addition to expanding our basic knowledge, a detailed understanding of these three phytohormones, involved in growth responses, stress tolerance, and fruit ripening, may provide insight for the eventual improvement of plants of agricultural or medicinal importance. For instance, determining how to increase stress tolerance (ABA perception) without altering growth characteristics (auxin perception) or ripening (ethylene perception) could be key to adapting plants to high stress areas (i.e., areas of low rainfall or saline soils).

The plant hormone auxin regulates plant growth and development to control crop yield. Indole-3-butyric acid, or IBA, is an inactive auxin precursor whose transport throughout the plant and conversion to the active hormone is critical to auxin function. Despite the great importance of IBA to plant growth, the mechanisms controlling IBA input into the level of available auxin are not well understood. This proposal aims to increase our understanding of how IBA-derived auxin impacts plant growth; this understanding will provide new tools to alter plant growth and development for use in crop improvement.

This CAREER proposal outlines experiments to elucidate developmental roles for indole-3-butyric acid (IBA) transport and IBA-derived auxin in the model plants Arabidopsis thaliana and Physcomitrella patens. Auxin is essential to plants, controlling cell division and cell expansion to orchestrate many developmental events and environmental responses. IBA is a biologically inactive molecule that is converted into the active auxin IAA (indole-3-acetic acid) by peroxisomal β-oxidation. IBA-derived auxin drives a wide range of seedling developmental processes, including cotyledon and root hair cell expansion, apical hook curvature, high-temperature-induced hypocotyl elongation, lateral root production, and root meristem maintenance. Similar to the active auxin IAA, the auxin precursor IBA is transported by carrier-mediated mechanisms.

The proposed experiments focus on understanding roles for IBA transport and IBA-derived auxin in plant development with four specific aims: 1) To determine the tissue-specificity of IBA-to-IAA conversion. 2) To identify components of IBA transport. 3) To characterize TRANSPORTER OF IBA (TOB) family regulation of IBA transport and plant development. 4) To compare the roles of IBA-derived auxin and IBA transport in the nonvascular plant Physcomitrella patens. The proposed studies will advance our understanding of auxin regulation of plant development.
This research will also support the creation of an inquiry-based laboratory to increase opportunities for undergraduate research at Washington U. This course addresses a critical need for a cell biology lab course in the curriculum; the primary objectives of this course are to increase student ability to form and test hypotheses and to raise student awareness of the diverse biological systems available for basic research. Aim 4 of this proposal will be directly integrated into the course and students will present their work at the WU Research Symposium at the end of the semester. In addition, this research will support the Missouri Local Auxin Meeting, co-organized by the PI to strengthen and expand the local auxin community, and additional K-12 outreach activities.

? DESCRIPTION (provided by applicant): Hormone-mediated modulation of gene activation or repression through transcription factors is central to all organisms. Auxin Response Factor (ARF) transcription factors are critical modulators of plant growth and provide an ideal model for exploring hormone control of gene activation and repression. We have recently identified protein multimerization and proteasomal degradation as two previously unknown mechanisms that regulate ARF activity. The long-term goal of this research project is to determine the importance of these ARF regulatory mechanisms in Arabidopsis thaliana transcriptional control. Elucidating the molecular mechanism of ARF regulation likely will uncover control processes common to other transcription factors. A repression-derepression paradigm regulates ARF activity. Under low concentrations of the hormone auxin, ARF transcriptional activity is repressed by Aux/IAA repressor proteins. When auxin concentrations increase, a co-receptor complex, comprised of an F-box protein (TIR1) and an Aux/IAA repressor protein, directly binds auxin. The F-box protein participates in a Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase, which targets the Aux/IAA protein for degradation. This degradation event relieves ARF repression, thereby allowing auxin-regulated gene transcription. Although this molecular model of repression and derepression for auxin activity appears relatively simple, our recent preliminary data suggest several exciting new control components and posttranslational modifiers influence ARF transcriptional activity and protein accumulation. This project aims to elucidate auxin signaling molecular mechanisms by identifying regulators of ARF protein activity and accumulation. To achieve this goal, we will use a combination of biochemical, biophysical, cell biology, synthetic biology, molecular and genetic techniques to gain insight into factors that influence ARF activity. The first aim is to understand the role of protein multimerization in the regulation of ARF transcriptional activity. Studies in both a synthetic yeast auxin response system and in planta will be used to test aspects of this aim, which includes functional assays and 3C analysis. The second aim is understand the role of multimerization in the regulation of ARF localization. We will determine whether ARF posttranslational modification affects ARF cellular localization. Our third aim is to establish rols for ARF proteasome-dependent degradation in regulating auxin response and plant development. We will use a variety of genetic, biochemical, and cell biology techniques to understand the biological and developmental roles for regulated ARF stability. The proposed research is innovative because our approaches focus strongly on the molecular understanding of ARF regulation, guided by our recent structural data on ARF7 and it has the potential to dramatically alter the auxin signaling model. The proposed research is significant because it is expected to advance and expand understanding of transcription factor regulation, using ARF factors as a model.