Purification and Partial Characterization of Tomato Extensin Peroxidase

Abstract

Early plant defense response is characterized by elevation of activity of peroxidases and enhanced insolubilization of hydroxyproline-rich glycoproteins, such as extensin, i n the cell wall.The insolubilization process (cross-linking between soluble extensin precursor molecules) is catalyzed by extensin peroxidases. We have ionically eluted extensin peroxidases from intact water-washed suspension-cultured tomato (hybrid of Lycopersicon esculenfum Mill.and Lycopersicon peruvianum L. [Mill.]) cells and purified them to homogeneity by molecular sieve and cation-exchange chromatography. Four ionic forms of peroxidase (PI, PII, EPIII, and EPIV) were resolved; only the latter two cross-linked tomato 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 investigated: soluble extensin and potato lectin (a hydroxyproline-rich glycoprotein with a domain that strongly resembles extensin) were crosslinked by only two forms of the enzyme, whereas bovine serum albumin, aldolase, insulin, a number of other marker proteins, and proteins eluted from tomato cells (except extensin) could not be cross-linked. We have also isolated a yeast elicitor that enhances total peroxidase activity and extensin insolubilization within 1 h of challenge i n cultured cells of tomato. A highly sensitive enzymelinked immunosorbent assay technique using polyclonal antiserum raised against soluble tomato extensin was used to demonstrate extensin insolubilization in vivo. A tomato cell-wall peroxidase that cross-links extensin has been purified and may have a role in plant defense.

Rapid Deposition of Extensin during the Elicitation of Grapevine Callus Cultures Is Specifically Catalyzed by a 40-Kilodalton Peroxidase

“…Confirming this, two of the internal peptide sequences obtained from GvEP could be mapped to conserved sequences in helixes B and J of the class III peroxidase structure (Schuller et al, 1996;Gajhede et al, 1997;Mirza et al, 1999). To date, the purification of only three other EPs has been reported (Brownleader et al, 1995;Schnabelrauch et al, 1996;Jackson et al, 1999) and none of these unusual peroxidases have been sequenced. Obtaining further peptide sequences of GvEP leading to the isolation of its cDNA clone therefore would be informative.…”

“…In the tomato system, at least two peroxidases have been demonstrated to have a high capacity for the oxidative cross-linking of extensin, whereas other tomato peroxidases (Brownleader et al, 1995) and several others derived from different species (Schnabelrauch et al, 1996) did not. A basic peroxidase with a similarly high capacity recently was purified from lupin, whereas an acidic form presented negligible activity against extensin (Jackson et al, 1999).…”

Di-isodityrosine Is the Intermolecular Cross-link of Isodityrosine-rich Extensin Analogs Cross-linked in Vitro

“…Although the pI 4.6 isozyme has a specific activity 2-3 orders of magnitude greater than a basic tomato peroxidase that crosslinks P1 extensin in vitro (23), its substrate range remains undefined. Conceivably, the cross-linking of multiple extensins might involve multiple extensin peroxidases, as several extensin peroxidases have been identified in a range of species (20,23,24,47). We therefore tested a P3 analog, YK20, as a substrate.…”

“…However, to date we lack direct demonstration of an extensin intermolecular cross-link. The results of extensin in vitro cross-linking assays have been difficult to interpret as the cross-linking substrates contained both Tyr and Lys in more than one type of repetitive motif, and the amino acids formed by cross-linking were not identified (20,23,24). Other approaches involving the isolation of intermolecularly crosslinked peptides from the cell wall itself have also proven intractable.…”

Differential Effect of Purified Spruce Chitinases and β-1,3-Glucanases on the Activity of Elicitors from Ectomycorrhizal Fungi

“…In these myc-mutants fungal strains of Glomus mosseae, which successfully formed arbuscular mycorrhizae on wild-type pea, failed to differentiate mycorrhizal structures because of the induction of physical barriers in the plant tissue located under the appressoria . It is known that elicitors can trigger processes such as lignification, callose apposition, and peroxidative crosslinking of extensin, which are the basis for an induced barrier formation in plant cell walls (Benhamou and Lafontaine, 1995;Brownleader et al, 1995). In this respect it was shown that elicitors released by the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull.…”