The Arabidopsis Vacuome - Towards A Virtual Vacuol

这个项目背后的概念将是编译一个虚拟植物液泡（“液泡组学”）数据库，以拟南芥为模型。在短期内，该项目的目标将是：(i)通过1-D和2-D电泳（2-DE）、同位素编码亲和标记（ICAT）、基质辅助激光解吸电离飞行时间（MALDI-TOF）和/或Q-Tof ms相结合的方法测定酿酒酵母的空泡腔内蛋白补体（ii）使用与步骤(i)类似的程序测定拟南芥的空泡腔内蛋白补体。如果最初一年的资助延长，这些研究将扩大和补充，包括：（iii）通过液相等电聚焦（IEF）和多维蛋白质鉴定技术（MudPIT）结合2-DE、ICAT、MALDI-TOF和/或Q-Tof ms来测定拟南芥液泡的液泡膜蛋白补体（iv）阐明拟南芥液泡的平均代谢物谱；(v)通过应用广泛的LC-MS-based分析程序，定义步骤（iv）中鉴定的液泡代谢物的摄取模式。（vi）通过在步骤（iv）中鉴定的化合物亚群，通过对步骤(i)中鉴定的同源物进行酵母敲除和/或通过蛋白质本身的异源表达，来阐明步骤（iii）中鉴定的液泡蛋白的功能能力。令人惊讶的是，我们对酶的机制知之甚少，除了氨基酸、糖、羧酸、储存蛋白质和无机离子之外，储存在液泡中的物质的类型，这些化合物是如何进入这个隔间的，以及它们在摄取后经历的修饰。这种基础知识的缺乏给许多代谢过程的合理分析和最终的植物代谢工程带来了问题。构建一个隔间工具箱，详细说明液泡汁液的酶和化学组成，以及后者如何通过液泡膜的运输建立和维持，将促进旨在理解植物生物学的基础研究，以及旨在提高植物营养质量的工程研究，用于提供药物或其前体，或用于环境修复应用。

The concept behind this project will be to compile a virtual plant vacuole ('vacuomics') database using Arabidopsis thaliana as the model. In the near term, the objectives of this project will be: (i) Determination of the vacuolar luminal protein complement of Saccharomyces cerevisiae by a combination of 1-D and 2-D electrophoresis (2-DE), isotope-coded affinity tagging (ICAT), matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and/or Q-Tof MS. (ii) Determination of the vacuolar luminal protein complement of Arabidopsis using procedures similar to those in step (i). If this initial one year's funding is extended, these investigations will be expanded and complemented to encompass: (iii) Determination of the vacuolar membrane protein complement of Arabidopsis vacuoles by the combination of liquid-phase isoelectric focusing (IEF) and multidimensional protein identification technology (MudPIT) with 2-DE and ICAT and MALDI-TOF and/or Q-Tof MS. (iv) Elucidation of the average metabolite profile of Arabidopsis vacuoles; (v) Definition of the modes of uptake of the vacuolar metabolites identified in step (iv) through the application of a broad-range LC-MS-based assay procedure. (vi) Elucidation of the functional capabilities of the vacuolar proteins identified in step (iii) via the subsets of compounds identified in step (iv) by the generation of yeast knockouts for their cognates identified in step (i) and/or by heterologous expression of the proteins themselves.It is surprising how little is known about the enzymic machinery, the types of substances, other than amino acids, sugars, carboxylic acids, storage proteins and inorganic ions, that are stored in the vacuole, how these compounds enter this compartment and the modifications they undergo after uptake. This lack of fundamental knowledge poses a problem for the rational analysis of many metabolic processes and ultimately plant metabolic engineering. Construction of a compartment toolbox detailing the enzymic and chemical composition of the vacuolar sap and how the latter is established and maintained by transport across the vacuolar membrane will facilitate basic research aimed at understanding plant biology and applied research aimed at engineering plants for enhanced nutritional quality, for the provision of pharmaceuticals or their precursors, or for environmental remediation applications.