RESUMEN
NO and H2O2 are important biological messengers in plants. They are formed during xylem differentiation in Zinnia elegans and apparently play important roles during the xylogenesis. To ascertain the responsiveness of the Z. elegans peroxidase (ZePrx) to these endogenous signals, the effects of NO and H2O2 on ZePrx were studied. The results showed that ZePrx is up-regulated by NO and H2O2, as confirmed by RT-qPCR, and that its promoter contains multiple copies of all the putative cis-elements (ACGT box, OCS box, OPAQ box, L1BX, MYCL box and W box) known to confer regulation by NO and H2O2. Like other OCS elements, the OCS element of ZePrx contains the sequence TACG that is recognized by OBF5, a highly conserved bZIP transcription factor, and the 10 bp sequence, ACAaTTTTGG, which is recognized by OBP1, a Dof domain protein that binds down-stream the OCS element. Furthermore, the ZePrx OCS element is flanked by two CCAAT-like boxes, and encloses one auxin-responsive ARFAT element and two GA3-responsive Pyr boxes. Results also showed that ZePrx may be described as the first protein to be up-regulated by NO and H2O2, whose mRNA contains several short-longevity conferring elements, such as a downstream (DST) sequence analogous to the DSTs contained in the highly unstable SAUR transcripts. The presence of these regulatory elements strongly suggests that ZePrx is finely regulated, as one may expect from an enzyme that catalyzes the last irreversible step of the formation of lignins, the major irreversible sink for the photosynthetically fixed CO2.
Asunto(s)
Asteraceae/enzimología , Peróxido de Hidrógeno/farmacología , Óxido Nítrico/farmacología , Peroxidasa/genética , Regiones Promotoras Genéticas/genética , Regiones no Traducidas 5'/genética , Asteraceae/efectos de los fármacos , Asteraceae/genética , Asteraceae/crecimiento & desarrollo , Secuencia de Bases , ADN Complementario/genética , ADN de Plantas/genética , Regulación Enzimológica de la Expresión Génica/genética , Regulación de la Expresión Génica de las Plantas/genética , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Lignina/análisis , Datos de Secuencia Molecular , Motivos de Nucleótidos , Peroxidasa/aislamiento & purificación , Peroxidasa/metabolismo , ARN de Planta/genética , Elementos de Respuesta/genética , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Alineación de Secuencia , Regulación hacia ArribaRESUMEN
The major basic peroxidase (ZePrx) from Zinnia elegans suspension cell cultures was purified and cloned. The purification resolved ZePrxs in two isoforms (ZePrx33.44 and ZePrx34.70), whose co-translational and post-translational modifications are characterized. Based on the N-terminal sequence obtained by Edman degradation of mature ZePxs, it may be expected that the immature polypeptides of ZePrxs contain a signal peptide (N-terminal pro-peptide) of 30 amino acids, which directs the polypeptide chains to the ER membrane. These immature polypeptides are co-translationally processed by proteolytic cleavage, and modeling studies of digestions suggested that the processing of the N-terminal pro-peptide of ZePrxs is performed by a peptidase from the SB clan (S8 family, subfamily A) of serine-type proteases. When the post-translational modifications of ZePrxs were characterized by trypsin digestion, and tryptic peptides were analyzed by reverse phase nano liquid chromatography (RP-nanoLC) coupled to MALDI-TOF MS, it was seen that, despite the presence in the primary structure of the protein of several (disulphide bridges, N-glycosylation, phosphorylation and N-myristoylation) potential post-translational modification sites, ZePrxs are only post-translationated modified by the formation of N-terminal pyroglutamate residues, disulphide bridges and N-glycosylation. Glycans of ZePrxs belong to three main types and conduce to the existence of at least ten different molecular isoforms. The first glycans belong to both low and high mannose-type glycans, with the growing structure Man(3-9)(GlcNAc)(2). Low mannose-type glycans, Man(3-4)(GlcNAc)(2), coexist with the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(GlcNAc)(2), in the G(3) and G(4 )sub-isoforms of ZePrx33.44. In ZePrx34.70, on the other hand, the complex-type biantennary glycan, Man(3)Xyl(1)Fuc(3)(GlcNAc)(5), and the truncated (paucimannosidic-type) glycan, Man(3)Xyl(1)Fuc(1)(GlcNAc)(2), appear to fill the two putative sites for N-glycosylation. Since the two N-glycosylation sites in ZePrxs are located in an immediately upstream loop region of helix F'' (close to the proximal histidine) and in helix F'' itself, and are flanked by positive-charged amino acids that produce an unusual positive-net surface electrostatic charge pattern, it may be expected that glycans not only affect reaction dynamics but may well participate in protein/cell wall interactions. These results emphasize the complexity of the ZePrx proteome and the difficulties involved in establishing any fine structure-function relationship.
Asunto(s)
Asteraceae/enzimología , Peroxidasa/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Asteraceae/genética , ADN Complementario/genética , Activación Enzimática/efectos de los fármacos , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peroxidasa/química , Peroxidasa/genética , Polisacáridos/metabolismo , Biosíntesis de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Electricidad Estática , Homología Estructural de Proteína , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
* The most distinctive variation in the monomer composition of lignins in vascular land plants is that found between the two main groups of seed plants. Thus, while gymnosperm lignins are typically composed of guaiacyl (G) units, angiosperm lignins are largely composed of similar levels of G and syringyl (S) units. * However, and contrary to what might be expected, peroxidases isolated from basal (Cycadales and Ginkgoales) and differentially evolved (Coniferales and Gnetales) gymnosperms are also able to oxidize S moieties, and this ability is independent of the presence or absence of S-type units in their lignins. * The results obtained led us to look at the protein database to search for homologies between gymnosperm peroxidases and true eudicot S-peroxidases, such as the Zinnia elegans peroxidase. * The findings showed that certain structural motifs characteristic of eudicot S-peroxidases (certain amino acid sequences and beta-sheet secondary structures) predate the gymnosperm-angiosperm divergence and the radiation of tracheophytes, since they are found not only in peroxidases from basal gymnosperms, ferns and lycopods, but also in peroxidases from the moss Physcomitrella patens (Bryopsida) and the liverwort Marchantia polymorpha (Marchantiopsida), which, as typical of bryophytes, do not have xylem tissue nor lignins.
Asunto(s)
Evolución Molecular , Peroxidasas/química , Filogenia , Proteínas de Plantas/química , Secuencias de Aminoácidos , Cycadopsida/química , Cycadopsida/enzimología , Cycadopsida/metabolismo , Helechos/enzimología , Helechos/genética , Lignina/metabolismo , Datos de Secuencia Molecular , Peroxidasas/análisis , Peroxidasas/clasificación , Proteínas de Plantas/análisis , Proteínas de Plantas/clasificación , Estructura Secundaria de Proteína , Alineación de Secuencia , Xilema/química , Xilema/enzimologíaRESUMEN
The last step of lignin biosynthesis in Zinnia elegans suspension cell cultures (SCCs) catalyzed by peroxidase (ZePrx) has been characterized. The k(3) values shown by ZePrx for the three monolignols revealed that sinapyl alcohol was the best substrate, and were proportional to their oxido/reduction potentials, signifying that these reactions are driven exclusively by redox thermodynamic forces. Feeding experiments demonstrate that cell wall lignification in SCCs is controlled by the rate of supply of H(2)O(2). The results also showed that sites for monolignol beta-O-4 cross-coupling in cell walls may be saturated, suggesting that the growth of the lineal lignin macromolecule is not infinite.
Asunto(s)
Asteraceae/enzimología , Lignina/biosíntesis , Peroxidasa/química , Proteínas de Plantas/química , Asteraceae/citología , Células Cultivadas , Peroxidasa/metabolismo , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
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 > or =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.
Asunto(s)
Asteraceae/enzimología , Asteraceae/metabolismo , Lignina/biosíntesis , Peroxidasa/genética , Peroxidasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Glicosilación , Focalización Isoeléctrica , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Peroxidasa/química , Peroxidasa/aislamiento & purificación , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , TemperaturaRESUMEN
Nitric oxide (NO) is currently regarded as a signal molecule involved in plant cell differentiation and programmed cell death. Here, we investigated NO production in the differentiating xylem of Zinnia elegans by confocal laser scanning microscopy to answer the question of whether NO is produced during xylem differentiation. Results showed that NO production was mainly located in both phloem and xylem regardless of the cell differentiation status. However, there was evidence for a spatial NO gradient inversely related to the degree of xylem differentiation and a protoplastic NO burst was associated with the single cell layer of pro-differentiating thin-walled xylem cells. Confirmation of these results was obtained using trans-differentiating Z. elegans mesophyll cells. In this system, the scavenging of NO by means of 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO) inhibits tracheary element differentiation but increases cell viability. These results suggest that plant cells, which are just predetermined to irreversibly trans-differentiate in xylem elements, show a burst in NO production, this burst being sustained as long as secondary cell wall synthesis and cell autolysis are in progress.