Cell wall-bound cationic and anionic class III isoperoxidases of pea root: biochemical characterization and function in root growth

Cell wall isolated from pea roots was used to separate and characterize two fractions possessing class III peroxidase activity: (i) ionically bound proteins and (ii) covalently bound proteins. Modified SDS–PAGE separated peroxidase isoforms by their apparent molecular weights: four bands of 56, 46, 44, and 41kDa were found in the ionically bound fraction (iPOD) and one band (70kDa) was resolved after treatment of the cell wall with cellulase and pectinase (cPOD). Isoelectric focusing (IEF) patterns for iPODs and cPODs were significantly different: five iPODs with highly cationic pI (9.5–9.2) were detected, whereas the nine cPODs were anionic with pI values between pH 3.7 and 5. iPODs and cPODs showed rather specific substrate affinity and different sensitivity to inhibitors, heat, and deglycosylation treatments. Peroxidase and oxidase activities and their IEF patterns for both fractions were determined in different zones along the root and in roots of different ages. New iPODs with pI 9.34 and 9.5 were induced with root growth, while the activity of cPODs was more related to the formation of the cell wall in non-elongating tissue. Treatment with auxin that inhibits root growth led to suppression of iPOD and induction of cPOD. A similar effect was obtained with the widely used elicitor, chitosan, which also induced cPODs with pI 5.3 and 5.7, which may be specifically related to pathogen defence. The differences reported here between biochemical properties of cPOD and iPOD and their differential induction during development and under specific treatments implicate that they are involved in specific and different physiological processes. Abbreviations: cPOD: covalently bound peroxidase DAB: 3,3'-diaminobenzidine DEPMPO: spin-trap (5-diethoxy-phosphoryl-5-methyl-1-pyrroline-n-oxide) EPR: electron paramagnetic resonance HRP: horseradish peroxidase IAA: indole-3-acetic acid HRP: horseradish peroxidase IEF: isoelectric focusing iPOD: ionically bound peroxidase NAA: naphthalene acetic acid PNGase F: peptide N-glycosidase F PR: pathogen-related SDS–PAGE: sodium dodecyl sulphate–polyacrylamide gel electrophoresis SHAM: salicylhydroxamic acid TMB: tetramethyl benzidine WGA: wheat germ agglutinin