Discovery via Proteomics of a Novel Cell Signalling Pathway in Plants Involving Extracellular ATP

Proteomic techniques were used to identify plant extracellular matrix (ECM) proteins. Among the many classical cell wall proteins with known biochemical activity that were identified, new proteins predicted from their open reading frame in the database were localised to the ECM. Putative protein kinases and ATP-binding proteins were also identified in these cell wallenriched protein fractions. Bio-informatic analysis of the primary sequences of the putative kinases and ATP-binding proteins confirmed their ECM localisation, prompting a search for extracellular phosphoproteins. After identification of phosphorylated proteins in the plant ECM, subsequent experiments confirmed the existence of extracellular ATP and led to the discovery that extracellular ATP is a repressor of cell death in plants. Furthermore, external ATP has emerged as a control point of pathogen elicitor-induced programmed cell death. The studies described here constitute an example of how proteomics can be a powerful tool to drive discovery of novel signalling and/or metabolic pathways through integration of systematic protein identification, bio- informatics, and downstream hypothesis testing.