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.