PURIFICATION AND PARTIAL CHARACTERIZATION OF TOMATO EXTENSIN PEROXIDASE

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich g lycoproteins, such as extensin, in the cell wall. The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensi n peroxidases from intact water-washed suspension-cultured tomato (hyb rid of Lycopersicon esculentum Mill. and Lycopersicon peruvianum L. [M ill.]) cells and purified them to homogeneity by molecular sieve and c ation-exchange chromatography. Four ionic forms of peroxidase (Pi, PII , EPIII, and EPIV) were resolved; only the latter two cross-linked tom ato soluble extensin. The molecular weight (34,000-37,000), amino acid composition, and isoelectric point (9.0) of the extensin peroxidases were determined. Substrate specificities of the enzymes were investiga ted: soluble extensin and potato lectin (a hydroxyproline-rich glycopr otein with a domain that strongly resembles extensin) were crosslinked by only two forms of the enzyme, whereas bovine serum albumin, aldola se, insulin, a number of other marker proteins, and proteins eluted fr om tomato cells (except extensin) could not be cross-linked. We have a lso isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge in cultured cell s of tomato. A highly sensitive enzyme-linked immunosorbent assay tech nique using polyclonal antiserum raised against soluble tomato extensi n was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and m ay have a role in plant defense.