Large-scale proteomic analysis of the grapevine leaf apoplastic fluid reveals mainly stress-related proteins and cell wall modifying enzymes

Abstract

BACKGROUND: The extracellular space or apoplast forms a path through the whole plant and acts as an interface with the environment. The apoplast is composed of plant cell wall and space within which apoplastic fluid provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Large-scale proteomic analysis reveals the protein content of the grapevine leaf apoplastic fluid and the free interactive proteome map considerably facilitates the study of the grapevine proteome. RESULTS: To obtain a snapshot of the grapevine apoplastic fluid proteome, a vacuum-infiltration-centrifugation method was optimized to collect the apoplastic fluid from non-challenged grapevine leaves. Soluble apoplastic protein patterns were then compared to whole leaf soluble protein profiles by 2D-PAGE analyses. Subsequent MALDI-TOF/TOF mass spectrometry of tryptically digested protein spots was used to identify proteins. This large-scale proteomic analysis established a well-defined proteomic map of whole leaf and leaf apoplastic soluble proteins, with 223 and 177 analyzed spots, respectively. All data arising from proteomic, MS and MS/MS analyses were deposited in the public database world-2DPAGE. Prediction tools revealed a high proportion of (i) classical secreted proteins but also of non-classical secreted proteins namely Leaderless Secreted Proteins (LSPs) in the apoplastic protein content and (ii) proteins potentially involved in stress reactions and/or in cell wall metabolism. CONCLUSIONS: This approach provides free online interactive reference maps annotating a large number of soluble proteins of the whole leaf and the apoplastic fluid of grapevine leaf. To our knowledge, this is the first detailed proteome study of grapevine apoplastic fluid providing a comprehensive overview of the most abundant proteins present in the apoplast of grapevine leaf that could be further characterized in order to elucidate their physiological function.