Identification and characterisation of Ca 2+-pectate binding peroxidases in Arabidopsis thaliana

The Arabidopsis genome encodes many secretory guaiacol peroxidases (class III plant peroxidases, EC 1.11.1.7). These higher plant enzymes are found either in the vacuole or in the apoplast, where several functions have been attributed to them. Their localisation within the cell wall matrix is most likely important for their activity. In the present work, a gel consisting of polygalacturonate chains cross-linked by Ca 2+ and embedded in polyacrylamide was used to separate proteins from Arabidopsis leaves having an affinity for the Ca 2+-mediated conformation of pectin. This chromatographic technique selected a small number of cationic isoperoxidases able to bind to Ca 2+-pectate but not to Ca 2+-alginate, a polyuronate gel similar to Ca 2+-pectate. This result suggested that some of the Arabidopsis peroxidases have an affinity for pectin in vivo. Such a property could allow them to be properly distributed within the cell wall network. In addition, eleven cDNAs encoding an Arabidopsis peroxidase were expressed in the baculovirus-insect cell system. The capacity of the resulting recombinant peroxidases to bind Ca 2+-pectate and Ca 2+-alginate was also assessed. It appeared that 3 of them exhibited a Ca 2+-pectate binding activity that was resistant to the action of NaCl. The binding of these recombinant peroxidases to Ca 2+-alginate was much weaker than to Ca 2+-pectate, confirming the specificity of the interaction with the pectic structure.