The Arabidopsis Vacuome - Towards A Virtual Vacuol

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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.