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

以离子形式吸收必需营养物质对植物生长至关重要。植物获得的所有离子中约有70%含有氮。因此从数量上说，氮是最重要的营养物。在农业中，氮主要来自施肥（100-250公斤每公顷）。化肥生产成本高，耗能大，造成大量的径流，对环境和健康产生巨大的负面影响。更好地了解植物的氮素获取可能有助于提高植物的氮素效率或优化施肥实践。养分获取需要在最大的氮吸收和最小的毒性之间取得良好的平衡。氮的两种主要形式是铵态氮和硝态氮。铵可引起毒性，因此需要严格控制细胞内的吸收和转化效率。铵转运体的分子性质几十年来一直不为人所知。PIs实验室与布鲁塞尔的B. andr<s:1>一起确定了铵转运蛋白家族（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对应物（恒河猴因子）。细菌和真菌amt具有受体、转运和传感双重功能蛋白的功能。amt整合了铵水平、细胞能量供应和铵同化前体的可用性等信息。pi实验室发现AMTs形成三聚体复合物，并受一种新的变构调节机制的影响，该机制涉及细胞质c端作为一个跨调节域。三种相似/相同蛋白质复合物的独特反式调节是由细胞外铵（以及潜在的其他因素）调节的，因此可能是通过微调铵积累来保护免受铵毒性的关键。考虑到细菌AMT对应物在信号传导中的作用，可以想象植物AMT也参与了传感和信号整合。该项目的目标将是揭示控制铵吸收的调节机制，其与碳和能量状态的整合，并确定有助于防止铵毒性的机制。该提案有五个具体目标：目标1使用下一代测序和磷蛋白组学来确定植物对铵暴露的反应。目的2将研究调节AMT1 c端在感知和调节中的作用。在Aim 3中，酵母将用于测试控制铵运输的调节系统。Aim 4将尝试开发用于铵和α -酮戊二酸的遗传编码Förster共振能量转移传感器（pi实验室首创的一项技术），并部署这种传感器来监测野生型和转运体突变体体内的铵。最后，Aim 5将与晶体学家合作，尝试生成细菌和植物amt的结构。该项目旨在确定变构控制机制和反馈回路作为植物营养信号整合的关键要素。该项目采用了最先进的技术（Nextgen测序和生物物理工具，即基因编码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式的可用性以及植物的氮状态的信息。它将为了解植物如何在空间和时间复杂的土壤系统中对氮形态的优先排序、土壤勘探和根结构、最佳生长的关键过程做出决定提供基础。更广泛的影响：预计获得的见解将与改善肥料的使用和防止农业和森林生态系统中的营养物质损害有关。该项目将为高中生、本科生和博士后提供培训，重点关注植物营养学、细胞生物学和生物物理学领域的少数群体。

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.