Collaborative Research: 2010 Arabidopsis: Cellular and Subcellular Resolution of the Tryptophan-Related Pathways

导致吲哚氨基酸色氨酸的途径是至关重要的，因为色氨酸是动物和人类饮食所必需的，并且因为吲哚化合物在植物和动物中用作细胞间信号。 此外，吲哚途径位于初级和次级代谢的界面，并在动态代谢过程中起节点的作用，可以预测单个细胞类型的代谢状态。该项目的目的是证明代谢物定量或分析方法（代谢组学）的潜力，以提供在单细胞水平上的细胞和代谢的影响，破坏参与色氨酸生物合成的基因的洞察力。这种方法将回答有关细胞之间代谢差异的重要问题，这些细胞彼此非常接近，但它们的起源，功能或最终命运不同。 该项目将利用在应用于细胞特异性转录物分析时有效的方法，即在特定细胞类型中用荧光蛋白标记的融合蛋白分选细胞，并将其应用于亚微米尺度的靶向代谢物分析。 色氨酸的生物合成是一个很好的代谢途径，这样的研究，不仅是因为该途径存在于初级和次级代谢之间的界面，而且还因为增加色氨酸水平是一个潜在的有价值的作物性状和动物和人类健康的代谢工程的目标。一个项目网站（http：www.auxin.org）将详细介绍该项目的具体目标和进展情况，包括代谢物特征数据。 更广泛的影响：该项目的主要目标是在组织和细胞水平上推进代谢组学（代谢物定量）的方法，使其作为功能基因组学工具箱的一部分，经常可用于更广泛的植物群落。 PI将举办一个研讨会，向更广泛的科学界传播代谢组学方法。 该项目将培养博士后研究员，他们将有能力继续发展和应用代谢组学作为基因组学工具。该项目将与正在开展的招募和培训代表性不足群体成员的活动相结合，这些活动满足了2010年项目的目标，因为人们认识到需要侧重于代谢物谱分析，以确定定位组的特征，即有关基因和蛋白质表达模式以及活植物器官、组织、细胞和/或亚细胞水平代谢物浓度和流量的信息。正如2010年项目中期评估所指出的，拟南芥小分子生物学的知识不仅是全面了解植物生物学的基础，而且在植物作为人类和动物的食物来源以及药物来源的背景下也是如此。 作为一种必需氨基酸，色氨酸的生物合成具有广泛的相关性，所开发的方法将允许研究植物中的任何代谢物网络。

The pathways leading to the indolic amino acid tryptophan are of critical importance because tryptophan is required in animal and human diets and because indolic compounds are used as intercellular signals in plants and animals. In addition, indolic pathways reside at the interface of primary and secondary metabolism and function as a node in dynamic metabolic processes that may be predictive of the metabolic status of individual cell types. The objective of this project is to demonstrate the potential of metabolite quantification or profiling methods (metabolomics) to provide insight at the single cell level into the cellular and metabolic consequences of disrupting genes involved in tryptophan biosynthesis. This approach will answer important questions about differences in metabolism between cells that are in close proximity to one another, but which differ in their origin, function or ultimate fate. The project will take advantage of approaches that have been effective when applied to cell-specific transcript profiling, namely sorting of cells with fluorescent protein-tagged fusion proteins in specific cell types, and will apply them to targeted metabolite profiling on a submicro-scale. Tryptophan biosynthesis is an excellent metabolic pathway for such studies not only because the pathway exists at the interface between primary and secondary metabolism, but also because increased tryptophan levels are a potentially valuable crop plant trait and a target for metabolic engineering for animal and human health. A project website (http://www.auxin.org) will detail the specific objectives and progress of the project, including metabolite profiling data. Mutant lines will be deposited at the ABRC.Broader Impacts: The major objective of this project is to advance the methodology of metabolomics (metabolite quantification) at the tissue and cellular levels to the point that it is routinely available to the broader plant community as part of the functional genomics toolbox. The PIs will develop a workshop to disseminate metabolomics methods to the broader scientific community. This project will train postdoctoral fellows, who will be well-positioned to continue the development and application of metabolomics as a genomics tool. The project will interface with on-going activities to recruit and train members of underrepresented groups.The activities meet the goals of the 2010 Project by addressing a recognized need to focus on metabolite profiling in order to characterize the localizome, that is, information about gene and protein expression patterns and metabolite concentrations and flux at the organ, tissue, cellular and/or subcellular levels in the living plant. As noted in the 2010 Project mid-course assessment, knowledge of small molecule biology in Arabidopsis is not only fundamental for a holistic understanding of plant biology, but also in the context of plants as a source of food for both humans and animals as well as sources of pharmaceuticals. As an essential amino acid, tryptophan biosynthesis is broadly relevant, and the approaches developed will allow the study of any metabolite network in plants.