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至关重要，因为动物和人类的饮食中都需要色氨酸，而且吲哚化合物在动植物中被用作细胞间信号。此外，吲哚通路位于初级和次级代谢的交界处，在动态代谢过程中起着节点的作用，可以预测单个细胞类型的代谢状态。本项目的目的是展示代谢物量化或图谱分析方法(代谢组学)的潜力，以便在单细胞水平上洞察参与色氨酸生物合成的干扰基因对细胞和代谢的影响。这种方法将回答有关彼此接近但在来源、功能或最终命运上不同的细胞之间新陈代谢差异的重要问题。该项目将利用在应用于特定细胞转录谱时有效的方法，即在特定细胞类型中用带有荧光蛋白标记的融合蛋白对细胞进行分选，并将它们应用于亚微米尺度的目标代谢物谱分析。色氨酸的生物合成对于这类研究来说是一个很好的代谢途径，不仅因为这条途径存在于初级和次级代谢之间，而且因为增加的色氨酸水平是一种潜在的有价值的作物性状和代谢工程的目标，以促进动物和人类的健康。项目网站(http://www.auxin.org))将详细说明项目的具体目标和进展，包括代谢物概况数据。突变系将存放在ABRC。Broader Impact：该项目的主要目标是推进组织和细胞水平的代谢组学(代谢物量化)方法，使其作为功能基因组学工具箱的一部分，经常可供更广泛的植物界使用。PIS将开发一个研讨会，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，他们将处于有利地位，继续开发和应用代谢组学作为基因组学工具。该项目将与正在进行的招募和培训代表性不足群体成员的活动相结合。拟议的活动满足了2010年项目的目标，满足了一个公认的需要，即侧重于代谢物图谱，以便确定本地化组的特征，即关于活植物器官、组织、细胞和/或亚细胞水平的基因和蛋白质表达模式以及代谢物浓度和通量的信息。正如2010年项目中期评估指出的那样，关于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。作为一种必需的氨基酸，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 proposed 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.