Purification and Partial Characterization of Extensin Peroxidase

The primary plant cell wall of higher plants is a focal point for growth regulation and disease resistance. Rapid responses to pathogen interaction are of fundamental importance because these fast reactions generate both primary components of plant resistance to pathogens and messenger systems that activate slower regulatory mechanisms mediated through gene activation and subsequent de novo protein synthesis.
Plants activate a large array of defence responses during microbial infection. These include the elevation of activity of peroxidases and enhancement of insolubilization of a hydroxyproline-rich glycoprotein (HRGP), called extensin, in the cell wall. This occurs within minutes after exposing plant cells to a pathogen or an elicitor derived from a pathogen or non-pathogen. Extensin insolubilization (cross-linking between preexisting soluble extensin precursor molecules) is catalyzed by a peroxidase referred to as 'extensin peroxidase’, in the presence of hydrogen peroxide and monitored by an in vitro Superose-12 FPLC assay.
Extensin and two ionic forms of 'extensin peroxidase' (Mono S EPIII and EPIV) were ionically eluted from intact water-washed tomato cells in suspension culture and purified to homogeneity. The molecular weights, amino acid composition and isoelectric points (pI) of both ionic forms of 'extensin peroxidase’ were demonstrated (Ahmed ef al., 1995). The substrate specificity of these enzymes has also been investigated. 'Extensin peroxidase’ is relatively substrate-specific (i.e. a particular enzyme will act primarily on a certain substrate) and appears to have a higher specificity towards its natural substrate extensin rather than other commercially purchased peroxidase substrates.
A yeast (Saccharomyces cerevisiae) derived elicitor known to be pathogenic to tomatoes has been purified. Preliminary studies have demonstrated that elicitation of tomato fruit with this yeast elicitor for 30 min enhances extensin insolubilization.