Rapid deposition of extensin during the elicitation of grapevine callus cultures is specifically catalyzed by a 40-kilodalton peroxidase

Elicitation or peroxide stimulation of grape (Vitis vinifera L. cv Touriga) vine callus cultures results in the rapid and selective in situ insolubili zation of an abundant and ionically bound cell wall protein-denominated GvP 1. Surface-enhanced laser desorption/ionization/time of flight-mass spectro metry analysis, the amino acid composition, and the N-terminal sequence of purified GvP1 identified it as an 89.9-kD extensin. Analysis of cell walls following the in situ insolubilization of GvP1 indicates large and specific increases in the major amino acids of GvP1 as compared with the amino acid s present in salt-eluted cell walls. We calculate that following deposition , covalently bound GvP1 contributes up to 4% to 5% of the cell wall dry wei ght. The deposition of GvP1 in situ requires peroxide and endogenous peroxi dase activity. Isoelectric focusing of saline eluates of callus revealed on ly a few basic peroxidases that were all isolated or purified to electropho retic homogeneity. In vitro and in situ assays of extensin cross-linking ac tivity using GvP1 and peroxidases showed that a 40-kD peroxidase cross-link ed GvP1 within minutes, whereas other grapevine peroxidases had no signific ant activity with GvP1. Internal peptide sequences indicated this extensin peroxidase (EP) is a member of the class III peroxidases. We conclude that we have identified and purified an EP from grapevine callus that is respons ible for the catalysis of GvP1 deposition in situ during elicitation. Our r esults suggest that GvP1 and this EP play an important combined role in gra pevine cell wall defense.