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

Abstract

The major basic peroxidase from Zinnia elegans (ZePrx) suspension cell cultures was purified and cloned, and its properties and organ expression were characterized. The ZePrx was composed of two isoforms with a M r (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M r of 33,440 (ZePrx33.44). Both isoforms showed absorption maxima at 403 (Soret band), 500, and 640 nm, suggesting that both are high-spin ferric secretory class III peroxidases. M r differences between them were due to the glycan moieties, and were confirmed from the total similarity of the N-terminal sequences (LSTTFYDTT) and by the 99.9% similarity of the tryptic fragment fingerprints obtained by reverse-phase nano-liquid chromatography. Four full-length cDNAs coding for these peroxidases were cloned. They only differ in the 5#-untranslated region. These differences probably indicate different ways in mRNA transport, stability, and regulation. According to the k cat and apparent K m RH values shown by both peroxidases for the three monolignols, sinapyl alcohol was the best substrate, the endwise polymerization of sinapyl alcohol by both ZePrxs yielding highly polymerized lignins with polymerization degrees $87. Western blots using anti-ZePrx34.70 IgGs showed that ZePrx33.44 was expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 was only expressed in roots and young hypocotyls. None of the ZePrx isoforms was significantly expressed in either leaves or cotyledons. A neighbor-joining tree constructed for the four full-length cDNAs suggests that the four putative paralogous genes encoding the four cDNAs result from duplication of a previously duplicated ancestral gene, as may be deduced from the conserved nature and conserved position of the introns.

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

“…Plant PRX proteins are involved in several important physiological and developmental processes, including lignin and suberin formation, the cross-linking of cell wall components, wound healing, the removal of H 2 O 2 , the oxidation of toxic reductants, and defense against pathogen or insect attack (Gabaldón et al, 2005;Bindschedler et al, 2006;Cosio and Dunand, 2009;Daudi et al, 2012). PRXs are encoded by a large gene family.…”

“…Based on the absence or presence of the C-terminal signal peptide, plant PRXs are divided into cell wall and vacuole types (Matsui et al, 2011). The wall-type PRXs are involved in cell wall metabolism including lignin polymerization, whereas the vacuole-type PRXs are involved in defense responses against abiotic and biotic stresses (Ostergaard et al, 2000;Li et al, 2003;Gabaldón et al, 2005;Bindschedler et al, 2006;Costa et al, 2008). This functional divergence between cell wall and vacuole PRXs makes this gene family a model system for exploring the contribution of protein subcellular relocalization to the expansion of the gene family.…”

Biological Role of Pigment Production for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

“…Another source of ROS comes from differentiating thin-walled xylem cells and particular nonlignifying xylem parenchyma cells. These cells are capable of sustained H 2 O 2 production, which is important for the cross-linking that occurs during the lignification process of developing xylem elements that can diffuse widely to neighboring xylem cells (3,18,19).…”

Molecular Cloning and Characterization of a Vacuolar Class III Peroxidase Involved in the Metabolism of Anticancer Alkaloids in Catharanthus roseus

“…Peroxidase isoenzymes are targeted by an N-terminal signal peptide to the secretory pathway and have either the cell wall or the vacuole as final destinations. Class III peroxidases have been mostly implicated in key processes determining the architecture and defense properties of the plant cell wall, like lignin and suberin biosynthesis and crosslinking reactions (Barceló et al, 2004;Bernards et al, 2004;Fry, 2004;Ralph et al, 2004;Gabaldó n et al, 2005). They have also been implicated in auxin catabolism, in secondary metabolism, in root elongation, in hydrogen peroxide scavenging and production, and they are thought to play important roles in stress resistance and adaptation (Yoshida et al, 2003;Díaz et al, 2004;Kristensen et al, 2004;Mika et al, 2004;Sang et al, 2004;Sottomayor et al, 2004;Passardi et al, 2005Passardi et al, , 2006.…”