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 41 kDa were found in the ionically bound fraction (iPOD) and one band (70 kDa) 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

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 agglutini

Class III peroxidases: Functions, localization and redox regulation of isoenzymes

Class III peroxidases (POXs; EC. 1.11.1.7), are secretory, multifunctional plant enzymes that catalyze the oxidation of a variety of substrates by hydrogen peroxide (H2O2). They show a remarkable diversity of isoenzymes, are encoded by a large number of paralogous genes, and are involved in a broad range of metabolic processes throughout plant growth and development. Peroxidases isoenzymes are located in the cell wall, apoplast and vacuole, and may be either soluble or ionically and covalently cell wall bound. They are involved in cell wall cross-linking and loosening, lignification and suberization, auxin catabolism and secondary metabolism. Due to their ability to control the levels of reactive oxygen species (ROS), POXs are efficient components of the antioxidative system induced in response to environmental stress, such as pathogen attack, metal excess, salinity, drought and high light intensity. In addition to the peroxidative function, POXs can catalyze H2O2 production in the oxidative cycle. Peroxidases are responsible either for cell elongation or cell wall stiffening, affecting carbon allocation, auxin level and redox homeostasis, which implicates their key role as being in the regulation of growth and defence under stress condition. This chapter will discuss novel insights into the functions of PODs with special emphasis on their localization, substrate specificity and the regulation of redox homeostasis