Extensin network formation in Vitis vinifera callus cells is an essential and causal event in rapid and H2O2-induced reduction in primary cell wall hydration

Hydrogen peroxide treatment of grapevine (Vitis vinifera cv. Touriga) callus cell walls was seen to induce a marked reduction in their hydration and thickness. An analysis of matrix proteins demonstrated this occurs with the insolubilisation of an abundant protein, GvP1, which displays a primary structure and post-translational modifications typical of dicotyledon extensins. The hydration of callus cell walls free from saline-soluble proteins did not change in response to H2O2, but fully regained this capacity after addition of extensin-rich saline extracts. To assay the specific contribution of GvP1 cross-linking and other wall matrix proteins to the reduction in hydration, GvP1 levels in cell walls were manipulated in vitro by binding selected fractions of extracellular proteins and their effect on wall hydration during H2O2 incubation assayed.This approach allowed us to conclude that a peroxidase-mediated formation of a covalently linked network of GvP1 is essential and causal in the reduction of grapevine callus wall hydration in response to H2O2. Importantly, this approach also indicated that extensin network effects on hydration was only partially irreversible and remained sensitive to changes in matrix charge. We discuss this mechanism and the importance of these changes to primary wall properties in the light of extensin distribution in dicotyledons.The central role that the primary cell wall plays in regulating extension growth, cell adhesion and cell morphology, requires a tight temporal-spatial regulation of its rheological properties, which are ultimately determined by matrix composition and structure. Most current primary cell wall models agree that the major wall polymers are bound to each other largely non-covalently, although physically intertwined [1,2]. In these models, hemicellulose is associated with cellulose through hydrogen bonding and physical entrapment, and pectins form a relatively mobile gel around the cellulose-hemicellulose network or b