Purification and characterization of a neutral peroxidase induced by rubbing tomato internodes

Abstract

Rubbing applied to a young tomato internode inhibited the elongation of this internode and increased soluble peroxidase activity. These morphological and biochemical changes were observed both at the site of rubbing (local response) and in the neighbouring internode (systemic response). The cellular, biochemical, and molecular mechanisms leading to inhibition of internode elongation are not fully understood. It was proposed that mechanical stimuli increased the oxidation of IAA, via the induction of specific peroxidases and stimulated the lignification processes. In order to gain more information about the role of these enzymes, analysis of changes in peroxidase activities were performed. Qualitative analysis of isoperoxidases, by means of native cathodic PAGE, showed four induced isoforms termed C1, C2, C3, and C4. The major isoform (C2) was purified to homogeneity and partially characterized. This isoform is probably unglycosylated, with a molecular mass of 36 kDa and a neutral pI of 7.1. The effects of pH and temperature on the activity were determined with guaiacol as electron donor. Optima were obtained at pH 5 and at a temperature of 55°C. The activity of the purified enzyme was not affected by Ca2+, Mg2+ and Mn2+ as was reported for some basic peroxidases. Analysis of substrate specificity revealed that this isoperoxidase acted on ABTS, o‐dianisidine, pyrogallol, guaiacol, coniferyl alcohol (monolignol) and IAA but not on syringaldazine. Activitiy of C2 isoperoxidase on coniferyl alcohol and IAA suggests a possible role of peroxidase C2 in inhibition of internode elongation, observed in rubbed plants, probably via an increase in lignification processes and regulation of IAA levels in internode tissues.

Properties of Guaiacol Peroxidase Activities Isolated from Corn Root Plasma Membranes

“…Calcium probably maintains the conformation of the proteins, whereas Mn 2ϩ could be involved in regulatory processes (Van Huystee et al, 1996). However, Loukili et al (1999) characterized plant peroxidases that were not influenced by these ions. Furthermore, Mn 2ϩ was not detectable in PM from corn roots (Lü thje et al, 1995).…”

“…Most peroxidases from plants and animals seemed to have high temperature optima and show high thermal stabilities (Bakardjieva et al, 1996;Madhavan and Naidu, 2000). Apoplastic, cytosolic, and soluble peroxidases of several plant tissues showed temperature optima between 30°C and 60°C, the most between 50°C and 60°C (Soda et al, 1991;Bakardjieva et al, 1996;Nair and Showalter, 1996;Bernards et al, 1999;Loukili et al, 1999). Due to the fact that pmPOX1 had a lower temperature optimum than pmPOX2, the latter enzyme seemed to be more stable (Fig.…”

Specific functions of individual class III peroxidase genes

“…Many authors have reported the appearance or disappearance of specific peroxidase isoforms during a particular process or in a particular localization (e.g. Loukili et al, 1999;Allison and Schultz, 2004). Nevertheless it is generally difficult to associate the band observed on an isoelectric focusing (IEF) gel with a particular protein mainly because protein purification is not straightforward.…”

Effect of pH and temperature on peroxidase and polyphenoloxidase activities of litchi pericarp

“…Peroxidase activity in the extracts was measured as described by Loukili et al (1999) using 24 mM guaiacol and 60 mM hydrogen peroxide in a spectrophotometer, recording changes in absorbance (470 nm). Polyphenoloxidase activity in the extracts was measured as proposed by Shin et al (1997) in a spectrophotometer as well, recording the changes in absorbance (410 nm), using 100 mM 4-methylcatechol as substrate and 0.2 M phosphate buffer pH 6.5.…”