Structural motifs of syringyl peroxidases predate not only the gymnosperm-angiosperm divergence but also the radiation of tracheophytes.

* 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.

The apoplastic antioxidant system in Prunus: response to long-term plum pox virus infection.

This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxymercuribenzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (class I APX) and pHMB-insensitive APX (class III APX), superoxide dismutase (SOD), peroxidase (POX), NADH-POX, and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in class I APX, POX, NADH-POX, and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. It is concluded that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plants.

Characterization of the last step of lignin biosynthesis in Zinnia elegans suspension cell cultures.

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.

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(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.

Changes in stem lignins (monomer composition and crosslinking) and peroxidase are related with the maintenance of leaf photosynthetic integrity during Verticillium wilt in Capsicum annuum.

• Verticillium dahliae is a soilborne pathogen that causes vascular wilt in pepper (Capsicum annuum var. annuum). Here we study to what extent changes in the lignification response of peppers condition tolerance of wilt. • For this, the quantum yield (ΦPSII ), the linear electron transport rate (ETR), and the lignification response (monomer composition and crosslinking) were studied in three C. annuum cultivars differing in degree of tolerance. • The results showed that in tolerant cultivars (Padrón and Yolo Wonder), both ΦPSII and ETR showed significantly higher levels at saturating photosynthetically active radiation values. This was not, however, the case for cv. Luesia, which showed a significant decrease in ΦPSII , ETR and nonphotochemical quenching values, suggesting that photochemical processes are strongly damaged in this cultivar as a consequence of the disease. The analysis of stem lignins in tolerant cultivars revealed that they were mainly composed of p-hydroxyphenyl units, while levels of ß-O-4-linked coniferyl and sinapyl alcohols were significantly lower. • It is concluded that through the observed changes in stem lignins (monomer composition and crosslinking) peppers retard, since they maintain leaf photosynthetic integrity, but do not stop (since wilt symptoms are not avoided) V. dahliae fungal hyphae penetration.

O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction.

The nature and specificity of the Wiesner test (phloroglucinol-HCl reagent) for the aromatic aldehyde fraction contained in lignins is studied. Phloroglucinol reacted in ethanol-hydrochloric acid with coniferyl aldehyde, sinapyl aldehyde, vanillin, and syringaldehyde to yield either pink pigments (in the case of hydroxycinnamyl aldehydes) or red-brown pigments (in the case of hydroxybenzaldehydes). However, coniferyl alcohol, sinapyl alcohol, and highly condensed dehydrogenation polymers derived from these cinnamyl alcohols and aldehydes did not react with phloroglucinol in ethanol-hydrochloric acid. The differences in the reactivity of phloroglucinol with hydroxycinnamyl aldehydes and their dehydrogenation polymers may be explained by the fact that, in the latter, the unsubstituted (alpha,beta-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the beta carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuumvar. annuum, Populus albaL., and Pinus halepensisL. demonstrated the presence of 4- O-linked hydroxycinnamyl aldehyde end groups and 4- O-linked 4-hydroxy-3-methoxy-benzaldehyde (vanillin) end groups in lignins. However, given the relatively low abundance of 4- O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4- O-linked hydroxycinnamyl aldehyde structures contained in lignins. Histochemical studies showed that these aldehyde structures are assembled, as in the case of coniferyl aldehyde, during the early stages of xylem cell wall lignification.

In situ characterization of a NO-sensitive peroxidase in the lignifying xylem of Zinnia elegans.

The lignifying xylem from Zinnia elegans stems gives an intense reaction with 3,3',5,5'-tetramethylbenzidine (TMB), a reagent previously reported to be specific for peroxidase/H2O2. However, the staining of lignifying xylem cells with TMB is apparently the result of two independent mechanisms: one, the catalase-sensitive (H2O2-dependent) peroxidase-mediated oxidation of TMB, and the other, the catalase-insensitive oxidation of TMB, probably mediated by xylem oxidases which are specific from lignifying tissues. The catalase-insensitive oxidation of TMB by the Z. elegans xylem was sensitive to sodium nitroprusside (SNP), a nitric oxide (NO)-releasing compound that, when used at 5.0 mM, is capable of sustaining NO concentrations of 6.1 &mgr;M in the aqueous phase. This effect of SNP was totally reversed by 150 &mgr;M 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO), an efficient NO scavenger in biological systems, so the above-mentioned effect must be ascribed to NO, and not to other nitrogen oxides. This response of the catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem was similar to that shown by a basic peroxidase isolated from the intercellular washing fluid, which showed TMB-oxidase activity, and which was also inhibited by 5 mM SNP, the effect of SNP also being reversed by 150 &mgr;M PTIO. These results suggest that peroxidase was the enzyme responsible for the NO-sensitive catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem. Further support for this statement was obtained from competitive inhibitor-dissected histochemistry, which showed that this stain responded to peroxidase-selective competitive inhibitors, such as ferulic acid and ferrocyanide, in a similar way to the Z. elegans basic peroxidase. From these results, we conclude that this NO-sensitive catalase-insensitive oxidation of TMB is apparently performed by the Z. elegans basic peroxidase, and that the regulation of this enzyme by NO may constitute an intrinsically programmed event during the differentiation and death of the xylem.

Comparative study of the products of the peroxidase-catalyzed and the polyphenoloxidase-catalyzed (+)-catechin oxidation. Their possible implications in strawberry (Fragaria x ananassa) browning reactions.

The peroxidase- and polyphenoloxidase-catalyzed oxidations of (+)-catechin yield several products showing different degrees of polymerization, which are apparently responsible for the pigment decay and the associated browning reaction that occurs in processed strawberry fruits and their derived foods. In this work, we have purified both peroxidase and polyphenoloxidase from Oso Grande cv. strawberry fruits, and comparatively analyzed the products of their enzyme-mediated (+)-catechin oxidation. The joint analysis by reversed-phase and size-exclusion HPLC of the (+)-catechin oxidation products obtained with both enzymes indicate that they were qualitatively the same: dehydrodicatechin B4, a (+)-catechin quinone methide, dehydrodicatechin A, a (+)-catechin trimer, and a (+)-catechin oligomer with polymerization degree equal to or greater than 5. The main quantitative differences between the oxidative reactions were the great amount of oligomer formed in the case of the polyphenoloxidase-mediated reaction and the low amount of (+)-catechin reacted in the case of the peroxidase-mediated reaction. One of the possible reasons for such low levels of (+)-catechin consumption in the case of the peroxidase-mediated reaction was the possible inhibition by products of the enzyme-catalyzed oxidation. In fact, the peroxidase-mediated (+)-catechin oxidation was differentially inhibited by dehydrodicatechin A, showing a competitive type inhibition and a k(I) of 6.4 microM. In light of these observations, these results suggest that brown polymer formation, estimated as oligomeric compounds resulting from (+)-catechin oxidation, in strawberries is mainly due to polyphenoloxidase, and although peroxidase also plays an important role, it is apparently auto-regulated by product (dehydrodicatechin A) inhibition.