Streamline Protocol for <i>Arabidopsis</i> Apoplastic Fluid Isolation Enables a Detailed Proteomic View of the Plant Extracellular Space

The apoplastic space surrounding plant cells, encompassing the cell wall matrix, extracellular spaces, and xylem, is one of the least understood compartments within plant tissues due to its lack of limiting membranes and its unavoidable damage upon tissue homogenization. Using a streamlined vacuum-infiltration/centrifugation protocol to enrich for the <i>Arabidopsis</i> apoplastic fluid (APF) combined with in-depth tandem mass spectrometry, we provide an improved view of its proteome that includes over 1500 proteins possibly assigned to this compartment with minimized cytosolic contamination. Included are large and varied collections of polypeptides associated with cell wall metabolism, oxido-reductase reactions, cell-cell signaling, proteolysis, and pathogen protection via basal defense pathways. While numerous apoplast proteins were predicted to house N-terminal signal peptide sequences that direct extracellular secretion, many did not, suggesting widespread use of non-classical export route(s). Among APF constituents are numerous pathogenesis-related proteins, glycosidases, aspartyl and subtilisin-type serine proteases, and the complement of subunits that assemble the core particle of the 26S proteasome. When this APF proteome is compared with those based on two prior isolation methods, a consensus collection of 338 polypeptides emerges that offers a comprehensive view of the core APF proteome that manages the cell wall and interfaces with the environment.