Nodulin-26 Intrinsic Proteins: Multifunctional Transporters of Water and Metabolites in Plant Symbioses and Stress Responses

主要固有蛋白(MIP)是一个古老的膜通道家族，它介导水和不带电荷的代谢物的选择性运输。这些转运蛋白在膜生理中发挥着广泛的作用，从哺乳动物的肾功能和液体分泌到植物和微生物的渗透调节、胁迫适应和营养吸收和运输。MIP家族在植物中的种类特别多样，反映了它们在调节植物生长发育中的水分关系和对环境渗透挑战的适应性反应中的重要性。然而，最近的证据表明，植物MIP的一个亚类，即结蛋白-26内在蛋白或NIPS，具有更广泛的作用。这些蛋白质以大豆根瘤蛋白26家族的原型通道命名，具有与其他MIP相似的总体结构，但显示出独特的孔结构特征，导致水以及关键植物代谢物(包括氨、硼和碳多元醇)的多功能运输行为。本项目的主要目标是研究NIPs的结构和功能，以及调节机制和它们在植物中的代谢作用。具体目标有三个方面。首先，我们将研究两个不同的NIP“孔家族”(NIP I和II蛋白)的结构和运输功能，它们在关键的孔选择性区域不同，表现出不同的运输特异性。其次，将研究大豆根瘤蛋白26与调节蛋白(蛋白激酶和14-3-3蛋白)以及氮同化酶谷氨酰胺合成酶相互作用的功能意义。谷氨酰胺合成酶与根瘤菌根瘤中形成氨通道的结瘤蛋白26相互作用的发现，对豆科植物-微生物共生的固氮/同化过程具有潜在的意义。此外，结合和招募胞浆蛋白和代谢酶的能力代表了MIP研究和调控的一个新的新兴兴趣。第三，以拟南芥蛋白AtNIP2；1为模型，研究NIPs在植物根系对低氧和淹水胁迫的代谢适应中的新功能。AtNIP2；1参与乳酸外排的能力将被作为一种潜在的机制来研究，以调节pH并预防代谢适应洪水和低氧胁迫期间的细胞质酸中毒。从基础设施的角度，该项目将有助于理解微生物与植物之间固氮和同化的分子基础，以及植物系统的逆境调节和代谢适应。这项工作还将有助于对植物分子印迹蛋白结构/功能关系的基本了解。广泛的影响和教育领域该项目将继续服务于更大范围的植物膜生物化学和生理学领域的研究生和博士后研究员的培训，并向田纳西大学的多名年轻本科生介绍现代植物分子生物学和生物化学研究。此外，该项目还将作为P.I.“S参与暑期计划和指导来自不同背景的田纳西州高中生”的基础，包括参与田纳西大学S州长理学院的活动，以及该大学的少数族裔招募活动，如JUMP(加入大学少数群体计划)。最后，该项目还将支持P.I.参与由文理学院赞助的一个新的暑期计划，为田纳西州中学教师提供暑期教育和实验室体验。

Major Intrinsic Proteins (MIPs) are an ancient family of membrane channels that mediate the selective transport of water and uncharged metabolites. These transporters play a myriad of roles in membrane physiology ranging from renal function and fluid secretion in mammals to osmoregulation, stress adaptation and nutrient uptake and transport in plants and microbes. The MIP family is particularly diverse in plants, reflecting their importance in regulating water relations in plant growth and development and adaptive responses to environmental osmotic challenge. However, recent evidence suggests a broader role for a subclass of plant MIPs known as Nodulin-26 intrinsic proteins or "NIPs". These proteins, named for the archetype channel of the family, soybean nodulin 26, have an overall structure similar to other MIPs, but show unique pore structural features that result in multifunctional transport behavior of water as well as critical plant metabolites including ammonia, boron and carbon polyols. The overarching goal of this project is to investigate the structure and function of NIPs as well as mechanisms of regulation and their metabolic roles in planta. The specific goals are three-fold. First, the structure and transport function of two distinct "pore familes" of NIPs (NIP I and II proteins) that differ in key pore selectivity regions and show different transport specificity will be investigated. Second, the functional significance of the interaction of soybean nodulin 26 with regulatory proteins (protein kinases and 14-3-3 proteins) and the nitrogen assimilatory enzyme glutamine synthetase, will be investigated. The finding that glutamine synthetase interacts with nodulin 26, which forms an ammonia channel in legume-rhizobia root nodules, is of potential significance to the nitrogen fixation/assimilation process of this plant-microbe symbiosis. Additionally, the ability to bind and recruit cytosolic proteins and metabolic enzymes represents a new emerging interest in MIP research and regulation in general. Third, to examine a new function of NIPs in metabolic adaptation to low oxygen and flooding stress in plant roots using the Arabidopsis protein AtNIP2;1 as a model. The ability of AtNIP2;1 to participate in lactic acid efflux will be investigated as a potential mechanism for pH regulation and prevention of cytosolic acidosis during metabolic adaptation to flooding and low oxygen stress. From the perspective of infrastructure, this project will contribute to the understanding of the molecular basis of nitrogen fixation and assimilation between microbes and plants, as well as stress regulation and metabolic adaptation of plant systems. The work will also contribute to the basic understanding of structure/function relationships of plant MIPs.Broader Impact and Educational OutreachThis project will continue to serve in the training of graduate students and postdoctoral fellows in the larger area of Plant Membrane Biochemistry and Physiology, as well as to introduce multiple young undergraduate scholars at the University of Tennessee to modern plant molecular biology and biochemistry research. In addition, this project will also serve as a foundation for the P.I.''s involvement in summer programs and mentoring of Tennessee High School students from diverse backgrounds, including participation in the Governor''s School for Sciences at the University of Tennessee, as well as in minority recruitment activities at the University such as JUMP (Join the University Minority Project). Finally, the project will also support the P.I.''s involvement in a new summer initiative sponsored by the College of Arts and Sciences providing summer educational and laboratory experiences for Tennessee Middle School Teachers.