Arabidopsis 2010: The role of nutrient sensing and signaling for ammonium nutrition in plants

以离子形式吸收必需的营养物质对植物的生长至关重要。植物获得的所有离子中约有70%含有氮。因此，从数量上讲，氮是最重要的营养物质。在农业中，氮主要来自化肥的施用(100-250公斤ha-1a-1)。化肥生产成本高、耗能大，并造成严重的径流，对环境和健康造成巨大的负面影响。更好地了解植物中氮素的获取可能有助于设计提高氮素效率的植物或优化施肥实践。获取养分需要在最大限度地吸收氮和最小化毒性之间取得良好的平衡。氮素的两种主要形态是铵和硝酸盐。铵可以引起毒性，因此需要严格控制细胞内的摄取和转化效率。几十年来，人们一直不知道铵离子转运体的分子性质。PIS实验室与布鲁塞尔的B.André一起确定了铵运输机家族(AMT/MEP/Rh)的创始成员。植物AMT有助于识别长期寻找的细菌和人类同行(恒河猴因子)。细菌和真菌的AMT具有转运体的功能，是介导运输和感知的双功能蛋白。AMTS综合了有关氨水平、细胞能量供应和氨同化前体可获得性的信息。PIS实验室发现，AMTS形成三聚体复合体，并受到一种新的变构调节机制的影响，该机制涉及胞质C-末端作为反式调节域。在一个由三个相似/相同的蛋白质组成的复合体中，独特的反式调节受到胞外铵(以及潜在的其他因素)的调节，因此可能是通过微调铵的积累来防止铵中毒的关键。考虑到细菌AMT对应物在信号传递中的作用，可以想象植物AMT也参与了传感和信号整合。该项目的目标将是解开控制铵吸收及其与碳和能源状况的整合的调节机制，并确定有助于防止铵毒性的机制。该提案有五个具体目标：AIM 1使用下一代测序和磷酸蛋白质组学来确定植物对氨暴露的反应。目的2将研究调节性AMT1 C末端在传感和调节中的作用。根据目标3，酵母将被用来测试控制铵运输的调控系统。AIM 4将尝试开发用于氨和α-酮戊二酸的遗传编码的Förster共振能量转移传感器(这是PIS实验室开创的一项技术)，并部署这种传感器来监测体内野生型和转运体突变体中的铵。最后，与结晶学家合作，AIM 5将尝试产生细菌和植物AMT的结构。该项目旨在确定变构控制机制和反馈环作为植物营养信号整合的关键要素。该项目应用了最先进的技术(NextGen测序和生物物理工具，即遗传编码的FRET传感器)。长期目标将是了解植物如何整合有关无机和有机氮形态可获得性以及植物氮素状况的信息。它将为了解植物如何在氮素形态的优先顺序、土壤探索和根构型方面做出决定提供基础，这些是在时空复杂的土壤系统中实现最佳生长的关键过程。广泛影响：所获得的见解有望与改善化肥使用和防止农业和森林生态系统中养分的破坏有关。该项目将为高中生、本科生和博士后提供培训，重点是植物营养、细胞生物学和生物物理学之间的少数民族。

The uptake of essential nutrients in the form of ions is critical for plant growth. Approximately 70% of all ions acquired by plants contain nitrogen. Thus quantitatively, nitrogen is the most important nutrient. In agriculture, nitrogen derives from mainly fertilizer application, (100-250 kg ha-1 a-1). Fertilizer production is costly, energy-consuming, and causes significant run-off with dramatic negative environmental and health impact. A better understanding of nitrogen acquisition in plants may help to engineer plants with improved nitrogen efficiency or optimize fertilization practice. Nutrient acquisition requires a fine balance between maximal nitrogen uptake and minimization of toxicity. The two dominant nitrogen forms are ammonium and nitrate. Ammonium can cause toxicity, thus requiring tight control over uptake and conversion efficacy inside cells. The molecular nature of ammonium transporters remained unknown for many decades. The PIs lab, together with B. André, Brussels, identified the founding members of the ammonium transporter family (AMT/Mep/Rh). Plant AMTs helped identifying the long sought-for bacterial and human counterparts (Rhesus factors). Bacterial and fungal AMTs have function as transceptors, dual function proteins mediating transport and sensing. AMTs integrate information on ammonium levels, cellular energy supply, and availability of precursors for assimilation of ammonium. The PIs lab found that AMTs form trimeric complexes and are subject to a novel allosteric regulatory mechanism involving the cytosolic C-terminus as a trans-regulatory domain. The unique trans-regulation in a complex of three similar/identical proteins is regulated by extracellular ammonium (and potentially other factors), and thus may be key to protecting against ammonium toxicity by fine-tuning ammonium accumulation. Given the role of the bacterial AMT counterparts in signaling, it is conceivable that plant AMTs are involved in sensing and signal integration as well. The objective of this project will be to unravel the regulatory mechanisms that control ammonium uptake, its integration with carbon- and energy status, and identify mechanisms that help protecting against ammonium toxicity. The proposal has five specific aims: Aim 1 uses Next Generation Sequencing and phosphoproteomics to identify plant responses to ammonium exposure. Aim 2 will study the role of the regulatory AMT1 C-terminus in sensing and regulation. Under Aim 3, yeast will be used to test for regulatory systems controlling ammonium transport. Aim 4 will attempt to develop genetically encoded Förster resonance energy transfer sensors for ammonium and alpha-ketoglutarate (a technology pioneered by the PIs lab), and to deploy such sensors to monitor ammonium in vivo in wild type and transporter mutants. Finally in collaboration with crystallographers, Aim 5, will attempt to generate structures of bacterial and plant AMTs.The project aims at identifying allosteric control mechanisms and feed-back loops as key elements of integration of signaling in plant nutrition. The project applies state of the art technology (Nextgen sequencing and biophysical tools, i.e. genetically encoded FRET sensors). The long-term goal will be to learn how plants integrate information on the availability of inorganic and organic nitrogen forms and the nitrogen status of the plant. It will provide a basis for learning how plants render decisions on prioritization of nitrogen forms, soil exploration and root architecture, key processes for optimal growth in a spatially and temporally complex soil system.Broader impacts: The insights gained are expected to be relevant to improvement of use of fertilizer and prevention of damage by nutrients in agricultural and forest ecosystems. The project will provide training for high school and undergraduate students and postdocs with an emphasis on minorities at the interface between plant nutrition, cell biology and biophysics.