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

本研究的重点是豆科植物中参与共生固氮的类结节蛋白(NIPs)。这一过程代表了将大气中的氮转化为还原的氨的主要途径，还原的氨是一种生物可用的氮形式。共生是通过根瘤菌侵染豆科植物根部，形成根瘤这一固氮器官而形成的。根瘤菌被封闭在一个专门的植物细胞器隔间--共生体中。共生体膜介导细菌共生体与植物寄主之间的代谢产物交换，是成功共生的关键。NIPS属于一个古老的通道蛋白家族，它调节水和代谢物在细胞膜上的流动。节点蛋白26(Nod26)是一种NIP，是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的作用。首先，作为氨的快速运输者，Nod26被认为是为了促进这种固定最终产物从共生体中释放出来。此外，Nod26与谷氨酰胺合成酶形成相互作用的复合体，谷氨酰胺合成酶是氮同化的关键酶。这种相互作用被认为是通过允许氨气快速融入无毒氨基酸谷氨酰胺来防止有毒水平的氨气积累。在第二个角色中，Nod26促进了水在共生体膜上的整体移动，并被认为是控制根瘤组织内氧气扩散的渗透调节网络的一部分。这提供了对固氮率的微调调节，以响应新陈代谢需要和环境压力。本项目的目的是研究Nod26在大豆和模式豆科植物紫花苜蓿中的多功能共生作用。具体目标是：1.确定Nod26水氨运输的结构基础及其与谷氨酰胺合成酶的相互作用；2.研究Nod26对水和氨的选择性调节机制，以及它在影响根瘤气体扩散和固定速率的环境线索(例如，土壤干旱和内涝)下的整体运输行为；以及3.利用Medicago可用的分子遗传学工具，研究Nod26对根瘤菌-豆科共生的建立和维持的影响。该项目应有助于深入了解豆科植物/根瘤菌共生固氮的分子基础及其调控。此外，由于NOD 26是NIP转运蛋白家族的结构原型，该项目将阐明NIP在其他植物膜系统中运输选择性和生物学功能差异的分子基础。更广泛的意义和重要性这个项目将作为多名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，该项目将支持和促进田纳西大学诺克斯维尔分校(UTK)的以下STEM倡议：1.P.I作为科学主任参与田纳西州青少年科学和人文研讨会(TJSHS)，这是一个全州范围的计划，旨在促进高中生的基础科学研究。TJSHS每年举行一次，包括一个为期三天的研讨会，在此期间，田纳西州最好的高中生将被召集在一起，并有机会展示他们的研究。2.参与大学前学者计划，旨在为来自田纳西州东部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。3.参与P.I.S实验室的女研究生，作为东京大学STEM推广项目的榜样和导师，该项目以中学女孩为对象，旨在鼓励她们参与科学。

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.