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

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[subscript r] (determined by matrix-assisted laser-desorption ionization time of flight)...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 2005-11, Vol.139 (3), p.1138-1154
Main Authors: GABALDON, Carlos, LOPEZ-SERRANO, Matias, PEDRENO, Maria A, ROS BARCELO, A
Format: Article
Language:English
Subjects:
5'-untranslated region
Alcohols
Amino Acid Sequence
amino acid sequences
Amino acids
Asteraceae - enzymology
Asteraceae - metabolism
Base Sequence
Biochemical Processes and Macromolecular Structures
Biological and medical sciences
biosynthesis
cell suspension culture
Cells, Cultured
Chromatography
Cloning, Molecular
Complementary DNA
cultured cells
DNA, Complementary - genetics
Enzymes
Fundamental and applied biological sciences. Psychology
Glycosylation
Hypocotyls
Isoelectric Focusing
Isoenzymes - chemistry
Isoenzymes - genetics
Isoenzymes - isolation & purification
Isoenzymes - metabolism
isozymes
Lignin
Lignin - biosynthesis
Mesophyll cells
Metabolism
Molecular Sequence Data
multigene family
nucleotide sequences
peroxidase
Peroxidase - chemistry
Peroxidase - genetics
Peroxidase - isolation & purification
Peroxidase - metabolism
plant biochemistry
plant genetics
Plant physiology and development
plant proteins
Plants
Proteins
sequence analysis
Sequence Analysis, DNA
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Substrate Specificity
Temperature
tissue distribution
Zinnia elegans
Zinnia violacea
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Summary: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[subscript r] (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a M[subscript 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[subscript 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[subscript cat] and apparent K[subscript m][superscript 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.
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.105.069674