Nodulin Intrinsic Proteins at the Plant-microbe Symbiotic Interface: Multifunctional Roles in Metabolite and Water Transport in Nitrogen Fixation and Stress Responses

该项目的重点是豆科植物中参与共生固氮的节蛋白样内在蛋白（NIPs）。 该过程代表了将大气氮转化为还原氨的主要途径，还原氨是氮的生物可用形式。 共生关系是通过根瘤菌土壤细菌感染豆科植物根部而建立的，导致固氮器官根瘤的形成。 根瘤菌被封闭在一个特殊的植物细胞器室，共生体。 共生体膜介导细菌共生体和植物宿主之间的代谢物交换，是成功共生的关键。 NIP属于一个古老的通道蛋白家族，介导水和代谢物穿过细胞膜的流动。 Nodulin 26（Nod 26）是一种NIP，构成共生体膜的主要蛋白质组分，在固氮共生体中具有两个重要作用。 首先，作为氨的快速转运蛋白，Nod 26被提议促进这种固定终产物从共生体的释放。 此外，Nod 26与谷氨酰胺合成酶形成相互作用复合物，谷氨酰胺合成酶是关键的氮同化酶。 这种相互作用被假定为通过允许其快速掺入无毒氨基酸谷氨酰胺来防止毒性水平的氨气的积累。 在第二个角色中，Nod 26促进了水穿过共生体膜的大量运动，并被认为是控制结节组织内氧气扩散的生物调节网络的一部分。 这提供了响应于代谢需要和环境压力的固氮速率的微调调节。 本项目旨在研究Nod 26在大豆和模式豆科植物蒺藜苜蓿中的多功能共生作用。 具体目标是：1.明确Nod 26的水氨转运及其与谷氨酰胺合成酶相互作用的结构基础; 2.研究调节Nod 26对水和氨的选择性的机制，以及其响应环境线索的整体运输行为（例如，土壤干旱和水涝），影响结核气体扩散和固定率; 3.利用苜蓿中的分子遗传学工具，研究Nod 26对根瘤菌-豆科植物共生关系的建立和维持的影响。 该项目将有助于对豆科植物/根瘤菌共生固氮的分子基础及其调控的基本见解。 此外，由于Nod 26是NIP转运蛋白家族的结构原型，该项目将揭示NIP在其他植物膜系统中转运选择性和生物功能差异的分子基础。 更广泛的意义和重要性该项目将作为多个研究生和本科生研究学者，以及博士后助理，在生物化学和分子生物学的基本方面的培训工具。 此外，该项目将提供支持，并促进在田纳西大学，诺克斯维尔（UTK）以下STEM倡议：1。作为田纳西州青少年科学与人文研讨会（TJSHS）的科学主任，该研讨会是一个旨在促进高中生基础科学研究的全州范围内的项目。 TJSHS每年举行一次，包括为期三天的研讨会，在此期间，来自田纳西州各地的最优秀的高中生被召集，并有机会展示他们的研究。 2.参与大学预科学者计划，旨在为来自东田纳西州的高中科学学者提供在UTK和橡树岭国家实验室的多学期研究经验。 3.女研究生参加P.I.作为UTK STEM推广项目的榜样和导师（“Gadget Girls Adventures in STEM”），该项目针对中学女生，旨在刺激她们参与科学。

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.