The focus of this project is on Nodulin-like Instrinsic Proteins (NIPs) involved in symbiotic nitrogen fixation in legumes. This process represents a major pathway for conversion of atmospheric nitrogen into reduced ammonia, which is a biologically usable form of nitrogen. The symbiosis is established by the infection of legume roots by rhizobia soil bacteria, resulting in the formation of the nitrogen-fixing organ, the root nodule. Rhizobia bacteria are enclosed within a specialized plant organellar compartment, the symbiosome. The symbiosome membrane mediates the exchange of metabolites between the bacterial symbiont and plant host, and is critical for a successful symbiosis. NIPs belong to an ancient family of channel proteins that mediate the flow of water and metabolites across cellular membranes. Nodulin 26 (Nod26) is a NIP that constitutes the major protein component of the symbiosome membrane and has two important roles in nitrogen-fixing symbioses. First, as a rapid transporter of ammonia, Nod26 is proposed to facilitate the release of this fixation end product from the symbiosome. Further, Nod26 forms interaction complexes with glutamine synthetase which is a critical nitrogen assimilation enzyme. This interaction is postulated to prevent accumulation of toxic levels of ammonia gas by allowing its rapid incorporation into the nontoxic amino acid glutamine. In a secondary role, Nod26 facilitates the bulk movement of water across the symbiosome membrane and is proposed to be part of an osmoregulatory network that controls the diffusion of oxygen gas within nodule tissues. This provides a finely tuned regulation of nitrogen fixation rates in response to metabolic need and environmental stresses. The aim of this project is to investigate the multifunctional symbiotic role of Nod26 in soybean and a model legume, Medicago truncatula. The specific objectives are: 1. To define the structural basis for water vs. ammonia transport of Nod26 and its interaction with glutamine synthetase; 2. Investigate mechanisms that regulate Nod26 selectivity for water and ammonia, and its overall transport behavior in response to environmental cues (e.g., soil drought and water logging) that affect nodule gas diffusion and fixation rates; and 3. Address the impact of Nod26 on the establishment and maintenance of the rhizobia-legume symbiosis by using the molecular genetics tools available in Medicago. This project should contribute fundamental insights into the molecular basis of symbiotic nitrogen fixation in legume/rhizobia associations as well as its regulation. In addition, since Nod 26 is the structural archetype of the NIP transporter family, the project will shed light on the molecular basis for differences in transport selectivity and biological functions of NIPs in other plant membrane systems.
Broader significance and importance This project will serve as a training vehicle for multiple graduate students and undergraduate student research scholars, as well as a postdoctoral associate, in fundamental aspects of Biochemistry and Molecular Biology. In addition, this project will provide support and promote the following STEM initiatives at the University of Tennessee, Knoxville (UTK): 1. The P.I's involvement as scientific director of the Tennessee Junior Science and Humanities Symposium (TJSHS), which is a state-wide program designed to foster basic science research by high school students. The TJSHS is held annually and involves a three day symposium during which the very best high school students from throughout the state of Tennessee are convened and are given an opportunity to present their research. 2. Involvement in the Pre-collegiate Scholars Program designed to provide high school science scholars from East Tennessee with multi-semester research experiences at UTK and the Oak Ridge National Laboratory. 3. Participation of female graduate students in the P.I.'s laboratory as role models and mentors for a STEM outreach project at UTK ('Gadget Girls Adventures in STEM') which targets middle school girls and is designed to stimulate their participation in the sciences.