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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Cloning and Molecular Characterization of the Basic Peroxidase Isoenzyme from Zinnia elegans, an Enzyme Involved in Lignin Biosynthesis^1,[w]

Department of Plant Biology, University of Murcia, E–30100 Murcia, Spain

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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: A. Ros Barceló (rosbarce{at}um.es).

^[w] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.069674.

^* Corresponding author; e-mail rosbarce{at}um.es; fax 34–968–363–963.

Received August 9, 2005; returned for revision September 1, 2005; accepted September 12, 2005.

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