Coniferyl alcohol oxidase activity of a cell-wall-located class III peroxidase

Abstract

Coniferyl alcohol oxidase activity was determined in cell walls from hypocotyls of the following species belonging to the family Asteraceae: Calendula officinalis, Callistephus sinensis, Cosmos bipinnanthus, Helianthus annuus, Helianthus debilis and Zinnia elegans. In all the cases studied, coniferyl alcohol oxidase activity was partially located ionically-bound to cell walls and resided in a basic peroxidase, the activity of which was stimulated by H 2 O 2 . This enzymatic activity was insensitive to freezing and was inactivated by high H 2 O 2 concentrations, as tested both in vitro and in situ by using purified cell wall fractions. The peroxidase with coniferyl alcohol oxidase activity was purified from Z. elegans hypocotyls until apparent homogeneity, as checked by SDS-PAGE. It showed a visible spectrum typical of a haem-containing high-spin ferric secretory (class III) plant peroxidase. Coniferyl alcohol oxidase activity of this basic peroxidase constitutes about 0.25% of the activity shown in the presence of H 2 O 2 . The significance of the coniferyl alcohol oxidase activity in vivo was studied in Z. elegans hypocotyls by means of histochemical tests, which revealed that it was located in the H 2 O 2 -producing lignifying xylem cells. The results obtained from the histochemical probes suggest that the coniferyl alcohol oxidase activity of this basic peroxidase is physiologically irrelevant in tissues that accumulate H 2 O 2 , as is the case of the lignifying xylem, where the peroxidase activity of the enzyme favorably competes with the oxidase activity of the enzyme.

H2O2 generation during the auto‐oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis

“…Seeds of Z. elegans were purchased from W.R. van der Schoot (Hillegom, The Netherlands). Seedlings were grown for 26 days in a greenhouse under daylight conditions at 25°C [8]. Fully grown (lignifying) hypocotyls were used for these studies.…”

“…Zinnia elegans is a seasonal‐cycle flowering plant belonging to the Asteraceae family (considered to be one of the most evolved families among dicotyledon angiosperms) and is frequently used as a model for lignification studies [7]. The cell wall of lignifying Z. elegans hypocotyls contains a basic peroxidase of high p I , which has been characterized as a high‐spin heme class III peroxidase (EC 1.11.1.7) [8]. This strongly basic peroxidase has been localized in the lignin‐forming xylem from Z. elegans hypocotyls by means of competitive inhibitor‐dissected histochemistry [9], and it is considered a marker of xylem element differentiation in Z. elegans cell cultures [10].…”

“…A characteristic of this basic peroxidase is that it shows coniferyl alcohol oxidase activity [8], i.e. an oxidase activity in the absence of H 2 O 2 .…”

Subcellular Relocalization and Positive Selection Play Key Roles in the Retention of Duplicate Genes ofPopulusClass III Peroxidase Family

“…PRX activity at 25°C with ascorbic acid, pyrogallol, and guaiacol were measured using the methods described by Kvaratskhelia et al (1997), activity with ferulic acid was measured as described by Sanchez et al (1996), activity with coniferyl alcohol was measured according to the method of Barceló and Aznar-Asensio (1999), and activity with sinapyl alcohol was measured as described by Barceló et al (2000). Protein concentrations were determined by measuring A 280 (Layne, 1957).…”

Cloning and Molecular Characterization of the Basic Peroxidase Isoenzyme from Zinnia elegans, an Enzyme Involved in Lignin Biosynthesis

“…Molecular studies of this basic peroxidase isoenzyme are strongly hampered by the difficulty of obtaining the protein in large amounts (Sato et al, 1995). Transdifferentiating Z. elegans mesophyll cell cultures, leaves, stems, hypocotyls, and even roots are a limited source of the protein since it is partially covalently bound to cell walls (Masuda et al, 1983;Sato et al, 1995;Ros Barceló and Aznar-Asensio, 1999). A promising source of this isoenzyme may be Z. elegans plant cell cultures since the protein, being located in cell walls, should be rapidly secreted to the culture medium.…”