The hormone auxin is essential for a large number of cellular and developmental processes in plants. Auxin achieves many of its actions by inducing the expression of a large number of genes to carry out the necessary functions. The Aux/IAA protein family normally suppresses auxin-responsive gene expression. An intermediate step in auxin induced gene expression is the degradation of these proteins through a ubiquitination/ 26S proteasome regulated process that depends on the interaction of several multi-component protein complexes. The intervening steps for this auxin response pathway are far from being understood. Prior studies revealed the contribution of the Rac/Rop GTPases in relating the auxin stimulus to the intracellular machinery for Aux/IAA degradation. The auxin induced Aux/IAA protein degradation process involves impromptu assembly of the degradation apparatus in response to the signal and is regulated by the presence of the Aux/IAA substrates. Observations from cell culture and whole plant studies suggest that auxin and substrates together induce formation of protein complexes that appear as protein bodies in the nucleus (referred to as nuclear protein bodies, NPBs). Substrates and components of SCFTIR1, CSN and 26S proteasome are recruited into and co-exist in these protein complexes, although the dynamics of their recruitment into and exit out of the NPBs remain to be elucidated. These NPBs are biologically active since the level of substrates has been observed to decline from within these structures in response to auxin treatment. The main focus of the proposed research is to examine the biochemical property of these NPBs, their composition and activity, and to examine the presence of high molecular weight protein complexes involved in auxin signaling, or other more amenable signaling pathways that are known to be regulated by ubiquitination and 26S proteasome-mediated proteolysis under endogenous conditions.
Broader Impact. Ubiquitination and 26S proteasome-regulated proteolysis underlies a large number of cellular and developmental processes in all eukaryotes, including cell division, growth and differentiation, and developmental pathways from embryogenesis to reproduction, senescence and programmed cell death. Malfunctioning of this system results in severe developmental problems, including debilitating neurological diseases and cancer in human and compromised ability to cope with stress and pathogens in plants. A regulated and dynamic process for assembly and disassembly of the molecular machinery to carry out degradation of selected proteins has been speculated. Information obtained will be important toward understanding auxin signaling, which in itself is a significant aspect of plant biology and represents a paradigm in plant cell signaling. The knowledge should also be applicable to other ubiquitination/26S proteasome regulated processes. The proposed research will provide training opportunities for postdocs, graduate students and undergraduates. The current group of undergraduates in the PIs laboratories includes four women and two men (including US citizens but natives of Afghanistan, Nepal and Ukraine); three of them are participating in the research related to the proposed studies. New students from a campus-wide NSF supported program for minority students (SPUR), will be recruited to start this summer.
Intellectual merit. Auxin is an extensively studied plant hormone because of its central importance to growth and development. This research project focused on understanding the molecular mechanism of how plants perceive auxin and mediate the signal to regulate cellular processes, such as gene expression, to carry out auxin’s function. A prominent auxin perception system is located in the nucleus; it regulates auxin-inducible gene expression by a system referred to as SCFTIR1 complex and the 26S proteasome, which together degrade a family of transcription factors that repress auxin-inducible gene expression. In this system auxin directly interacts with SCFTIR1 and stimulates degradation of these repressors, releasing auxin-inducible gene from transcriptional repression. We discovered that a cytoplasmic signaling pathway, centered on a family of proteins, referred to as RAC/ROPs, also mediates auxin. RAC/ROPS are small GTPases and major cell membrane-located molecular switches mediating many exogenous signals to multiple intracellular pathways. Our earlier results, based largely on microscopic studies, showed that auxin and RAC/ROPs mediate the assembly of the SCFTIR1 and 26S proteasome complexes in the nucleus and that the transcription repressors are degraded inside these proteolytically active nuclear bodies. In this research project, we showed by biochemical approaches that these nuclear protein bodies are assemblies of the transcriptional repressors and all of the major components of the SCFTIR1 complex and 26S proteasome tested. Our results also showed that a similar strategy is utilized for the signaling of several other hormones besides auxin suggesting a broadly adopted mechanism. An important implication of our result, in particular because RAC/ROPs act from the cell membrane level, is that these small GTPases may act as intermediaries for a cell surface auxin perception system that has been elusive for identification. Our project identified several interacting proteins for RAC/ROPs; one of these was determined to be a cell surface- located receptor kinase called FERONIA, with the potential to perceive extracellular signals, such as auxin, and regulate intracellular pathways by phosphorylation modification of target proteins via its kinase domain. We showed that FERONIA regulates auxin-regulated root hair development via its interaction with the RAC/ROP signaling apparatus. We also identified another cell surface molecule known as LLG1 which interacts with FERONIA and potentially together they act as co-regulators of RAC/ROP-mediated signaling. FERONIA and LLG1 are novel regulatory factors for RAC/ROPs and potentially a cell surface auxin-signaling apparatus. As such, they also provide important leads to further dissect auxin and RAC/ROP signaling mechanisms. Broader impacts. With auxin being a key plant growth regulator and 26S proteasome being an almost universally used regulatory system controlling many important cellular and developmental processes, results from this project will have broad implications on basic biological knowledge. As such, the research reagents we generated from this project are of interest to a broad community and have been provided to interested colleagues. This project also provides the opportunities for training young scholars at various stages of preparation for a career in science. Together, two postdocs, two graduate students and two undergraduates have received extensive research training from this project. Four of these were women, one undergraduate has gone on to dental school, and another is current seeking employment in the biological research sector. Four additional undergraduates, two from under-represented groups, also received training from this project. One is currently seeking entry to a medical school, the other remains as a research undergraduate in the lab. We have also used results from this project in Freshman Seminars whose purpose was to interest incoming students to the wonders of biological research, irrespective of their scientific inclination. Participants in this project have also contributed materials generated from this project to "show and tell" projects at the Boston Museum of Science, reaching a very young student population.
