Class III peroxidases in plant defence reactions.

When plants are attacked by pathogens, they defend themselves with an arsenal of defence mechanisms, both passive and active. The active defence responses, which require de novo protein synthesis, are regulated through a complex and interconnected network of signalling pathways that mainly involve three molecules, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), and which results in the synthesis of pathogenesis-related (PR) proteins. Microbe or elicitor-induced signal transduction pathways lead to (i) the reinforcement of cell walls and lignification, (ii) the production of antimicrobial metabolites (phytoalexins), and (iii) the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). Among the proteins induced during the host plant defence, class III plant peroxidases (EC 1.11.1.7; hydrogen donor: H(2)O(2) oxidoreductase, Prxs) are well known. They belong to a large multigene family, and participate in a broad range of physiological processes, such as lignin and suberin formation, cross-linking of cell wall components, and synthesis of phytoalexins, or participate in the metabolism of ROS and RNS, both switching on the hypersensitive response (HR), a form of programmed host cell death at the infection site associated with limited pathogen development. The present review focuses on these plant defence reactions in which Prxs are directly or indirectly involved, and ends with the signalling pathways, which regulate Prx gene expression during plant defence. How they are integrated within the complex network of defence responses of any host plant cell will be the cornerstone of future research.

Lignification in plant cell walls.

Cell wall lignification is a complex process occurring exclusively in higher plants; its main function is to strengthen the plant vascular body. This process involves the deposition of ill-defined phenolic polymers, the so-called lignins, on the extracellular polysaccharidic matrix. These polymers arise from the oxidative coupling of three cinnamyl alcohols in a nonrandom reaction, in which cell wall polysaccharides appear to influence the freedom of cinnamyl alcohol radicals, giving rise to a highly orchestrated process. This review is focused on the most recent advances in the chemical, biochemical, cytological, physiological, and evolutive aspects of cell wall lignification. As we shall see throughout this review, there are still some open questions to be answered which may serve as the basis of future endeavors.

H(2)O(2) generation during the auto-oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis.

Characterization of lignified Zinnia elegans hypocotyls by both alkaline nitrobenzene oxidation and thioacidolysis reveals that coniferyl alcohol units are mainly found as part of 4-O-linked end groups and aryl-glycerol-beta-aryl ether (beta-O-4) structures. Z. elegans hypocotyls also contain a basic peroxidase (EC 1.11.1.7) capable of oxidizing coniferyl alcohol in the absence of H(2)O(2). Results showed that the oxidase activity of the Z. elegans basic peroxidase is stimulated by superoxide dismutase, and inhibited by catalase and anaerobic conditions. Results also showed that the oxidase activity of this peroxidase is due to an evolutionarily gained optimal adaptation of the enzyme to the microM H(2)O(2) concentrations generated during the auto-oxidation of coniferyl alcohol, the stoichiometry of the chemical reaction (mol coniferyl alcohol auto-oxidized/mol H(2)O(2) formed) being 0.496. These results therefore suggest that the H(2)O(2) generated during the auto-oxidation of coniferyl alcohol is the main factor that drives the unusual oxidase activity of this highly conserved lignin-synthesizing class III peroxidase.

Purification and characterization of alpha-3',4'-anhydrovinblastine synthase (peroxidase-like) from Catharanthus roseus (L.) G. Don.

An H2O2-dependent enzyme capable of coupling catharanthine and vindoline into alpha-3',4'-anhydrovinblastine (AVLB) was purified to apparent homogeneity from Catharanthus roseus leaves. The enzyme shows a specific AVLB synthase activity of 1.8 nkat/mg, and a molecular weight of 45.40 kDa (SDS-PAGE). In addition to AVLB synthase activity, the purified enzyme shows peroxidase activity, and the VIS spectrum of the protein presents maxima at 404, 501 and 633 nm, indicating that it is a high spin ferric heme protein, belonging to the plant peroxidase superfamily. Kinetic studies revealed that both catharanthine and vindoline were substrates of the enzyme, AVLB being the major coupling product.

Oxidation of hydroquinone by both cellular and extracellular grapevine peroxidase fractions.

The oxidation of hydroquinone by two peroxidase (EC 1.11.1.7) fractions obtained from the cells and spent medium of cell cultures of grapevine (Vitis vinifera cv Monastrell) has been studied, and their comparative efficacy (kcat/KM ratio) studied in both the H2O2-consuming and hydroquinone-consuming reactions. While the efficacy in the H2O2-consuming reaction is practically identical for both enzyme fractions, the cellular peroxidase has five-fold more efficacy in the hydroquinone-consuming reaction than the peroxidase located in the spent medium. Screening of cellular peroxidases capable of oxidizing hydroquinone on polyacrylamide gels, by means of a staining reaction based on the nucleophilic attack of 4-aminoantipyrine on p-benzoquinone in acidic media, reveals that all the cellular peroxidase isoenzymes are capable of oxidizing hydroquinone, probably yielding a quinone-diimine as a product of the staining reaction. Since isoperoxidases found in cellular fractions are also present in the spent medium, the values found for the different efficacies in the hydroquinone-consuming reaction must be considered as the results of the different proportions in which each peroxidase isoenzyme was found in the two fractions. The localization of a benzoquinone-generating system of high efficacy inside the plant cell, and probably located in vacuoles, is discussed with respect to the harmful role which the quinone/semiquinone pair might play in cell death, as part of the hypersensitive response expressed within the mechanism of plant disease resistance.

A spectrophotometric assay for quantitative analysis of the oxidation of 4-hydroxystilbene by peroxidase-H2O2 systems.

A new and sensitive spectrophotometric assay has been developed to study and to characterize kinetically the oxidation of 4-hydroxystilbene (an analogue of the putative viniferin precursor, trans-resveratrol, directly involved in the resistance mechanism of the grapevine to fungal diseases) by peroxidase-H2O2 systems. The technique measures the overall increase in absorbance at 248 nm in the reaction media, probably due to the formation of a phenoxy radical of the 4-hydroxystilbene (4-HS). This technique was developed by using the purified isoenzyme C of horseradish peroxidase and all the validity criteria (sensitivity and reproducibility) were checked. The results show that it is especially suitable for low activity measurements. It was finally applied to the determination of the oxidation rate of 4-HS by peroxidases isolated from the media of suspension-cultured grapevine cells, at two different developmental stages.

H(2)O(2)-mediated pigment decay in strawberry as a model system for studying color alterations in processed plant foods.

Phenolic and pigment (anthocyanin) stability in processing-ripe strawberries in response to aging under mildly oxidizing conditions, provoked by exogenous application of H(2)O(2), has been studied to design a simplified model system to study color alterations (pigment decay) that occur in strawberry-derived foods during processing and storage. The results showed that phenolic metabolism in strawberry slices during aging under mildly oxidizing conditions may be either oxidative (independent of exogenous H(2)O(2)) or peroxidative (dependent on exogenous H(2)O(2)), and that feeding strawberry slices with H(2)O(2) stimulates the oxidative phenomena which take place in their absence, such as the processes of anthocyanin and catechin degradation. The results also showed that because both (+)-catechin and anthocyanin levels in strawberry slices fall as H(2)O(2) increases, both p-hydroxybenzoic acid and brown polymeric compounds are formed. Comparison of these results with controls in the absence of H(2)O(2) suggests that peroxidase may play an important role in catechin consumption and in anthocyanin degradation and brown polymer formation during the aging of strawberry slices under mildly oxidizing conditions.

Cytochemical localization of phenol-oxidizing enzymes in lignifying Coleus blumei stems.

The cytochemical localization of the phenol oxidases, laccase and peroxidase, has been studied in pro-lignifying and lignifying Coleus blumei stem sections using 4-methoxy-alpha-naphthol as substrate. The results illustrated that, for short incubation times, both pro-lignifying and lignifying Coleus sections showed H2O2-dependent phenol oxidase (peroxidase-like) activity in epidermal and vascular tissues, while no detectable H2O2-independent phenol oxidase (laccase-like) activity was found in Coleus tissues. For long incubation times, H2O2-independent phenol-oxidases can also be detected in these tissues, however, this is probably due to the partial capability of intercellular washing fluid Coleus peroxidase to oxidize 4-methoxy-alpha-naphthol in the absence of exogenously added H2O2. This illustrates not only the importance of the substrate used, but also the importance of the incubation time, in the cytochemical localization of phenol oxidizing enzymes.

Effect of fosetyl-A1 on peroxidase from grapevine (Vitis vinifera) cells.

Grapevine (Vitis vinifera cv. Monastrell) suspension cell cultures were treated with fosetyl-A1, a widely used systemic fungicide for grapevine diseases caused by oomycetes, and examined at the electron microscope level for peroxidase cytochemistry. The results showed that treatment with fosetyl-A1 provokes an activation of both vacuolar sap and tonoplast-located peroxidase, already described as due to the basic peroxidase isoenzyme, B5, which was previously characterized as a constitutive marker of disease resistance against Plasmopara viticola in axillary bud cultures of Vitis spp. This activation of peroxidase isoenzyme B5, as seen at the electron microscope level, was confirmed by cytophotometric methods, but is in contrast with the unchanged enzyme level determined by biochemical methods. These results suggest a metabolic activation of peroxidase isoenzyme B5 as a consequence of fosetyl-A1 treatment, probably due to an acidification of the vacuole. This response was accompanied by the appearance of myelin-like structures inside the cytoplasm and osmiophylic-bodies inside the mitochondria. However, the latter structural changes cannot easily be related to the above described specific peroxidase response.

Distribution of lignin monomers and the evolution of lignification among lower plants.

Through application of chemical, biochemical and histochemical analyses, we provide new data on the absence/presence of syringyl lignins in the algal species Mastocarpus stellatus, Cystoseira baccata and Ulva rigida, the bryophytes Physcomitrella patens and Marchantia polymorpha, the lycophytes Selaginella martensii, Isoetes fluitans and Isoetes histrix, the sphenophyte Equisetum telmateia, the ferns Ceratopteris thalictroides, Ceratopteris cornuta, Pteridium aquilinum, Phyllitis scolopendrium and Dryopteris affinis, and the angiosperm Posidonia oceanica. Lignins, and especially syringyl lignins, are distributed from non-vascular basal land plants, such as liverworts, to lycopods and ferns. This distribution, along with the already reported presence of syringyl lignins in ginkgoopsids, suggests that syringyl lignin is a primitive character in land plant evolution. Here, we discuss whether the pathway for sinapyl alcohol recruitment was iterative during the evolution of land plants or, alternatively, was incorporated into the earliest land plants and subsequently repressed in several basal liverworts, lycopods, equisetopsids and ferns. This last hypothesis, which is supported by recent studies of transcriptional regulation of the biosynthesis of lignins, implies that lignification originated as a developmental enabler in the peripheral tissues of protracheophytes and would only later have been co-opted for the strengthening of tracheids in eutracheophytes.

Two distinct cell sources of H2O2 in the lignifying Zinnia elegans cell culture system.

The use of transdifferentiating Zinnia elegans mesophyll cells has proved useful in investigations of the process of xylem differentiation from cambial derivatives. Cultured mesophyll cells can be induced by external stimuli to proceed through temporally controlled developmental programs which conclude in the formation of single-cell-derived dead vascular tracheids and parenchyma-like elements. However, there is a gap in our knowledge concerning the role played by reactive oxygen species (O(2) (-) and H(2)O(2)) in the development of these vascular elements. In this study, we show by the following four independent and highly selective methods that transdifferentiating Z. elegans mesophyll cells are capable of producing reactive oxygen species: the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, which monitors O(2) (-) production, and the xylenol orange, 2,7-dichlorofluorescein diacetate, and CeCl(3) assays, which monitor H(2)O(2) production and localization. The joint use of these biochemical (XTT and xylenol orange) assays and cytochemical (2,7-dichlorofluorescein diacetate and CeCl(3)) probes revealed that transdifferentiating Z. elegans mesophyll cells do not show an oxidative burst but live in a strongly oxidative state during the entire culture period. In this state, H(2)O(2) is produced by both tracheary and parenchyma-like elements, the nonlignifying parenchyma-like cells acting quantitatively as the main source. The existence of these two sources of H(2)O(2) in this in vitro cell culture system may be especially relevant during the later stages of tracheary cell wall lignification, in which lignifying tracheary elements become hollow. In the case of differentiating tracheary elements, H(2)O(2) was located in the same place and at the same time as the onset of tracheary element lignification, i.e., at the primary cell wall during secondary thickening, supporting the view that the H(2)O(2) produced by this in vitro culture system is destined for use during lignin biosynthesis.

Xylem parenchyma cells deliver the H2O2 necessary for lignification in differentiating xylem vessels.

Lignification in Zinnia elegans L. stems is characterized by a burst in the production of H(2)O(2), the apparent fate of which is to be used by xylem peroxidases for the polymerization of p-hydroxycinnamyl alcohols into lignins. A search for the sites of H(2)O(2) production in the differentiating xylem of Z. elegans stems by the simultaneous use of optical (bright field, polarized light and epi-polarization) and electron-microscope tools revealed that H(2)O(2) is produced on the outer-face of the plasma membrane of both differentiating (living) thin-walled xylem cells and particular (non-lignifying) xylem parenchyma cells. From the production sites it diffuses to the differentiating (secondary cell wall-forming) and differentiated lignifying xylem vessels. H(2)O(2) diffusion occurs mainly through the continuous cell wall space. Both the experimental data and the theoretical calculations suggest that H(2)O(2 )diffusion from the sites of production might not limit the rate of xylem cell wall lignification. It can be concluded that H(2)O(2) is produced at the plasma membrane in differentiating (living) thin-walled xylem cells and xylem parenchyma cells associated to xylem vessels, and that it diffuses to adjacent secondary lignifying xylem vessels. The results strongly indicate that non-lignifying xylem parenchyma cells are the source of the H(2)O(2) necessary for the polymerization of cinnamyl alcohols in the secondary cell wall of lignifying xylem vessels.

Nitric oxide production by the differentiating xylem of Zinnia elegans.

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.

Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of alpha-3',4'-anhydrovinblastine: a specific role for a multifunctional enzyme.

We have characterized a basic peroxidase with alpha-3',4'-anhydrovinblastine (AVLB) synthase activity, which was purified from Catharanthus roseus leaves. This enzyme was the single peroxidase isoenzyme detected in C. roseus leaves, and the single AVLB synthase activity detected in C. roseus extracts. It was observed that the monomeric substrates of AVLB, vindoline and catharanthine, are both suitable electron donors for the oxidizing intermediates of the basic peroxidase, compounds I and II. Results also showed that the reaction proceeds by a radical-propagated mechanism. Substrate specificity studies of the enzyme revealed that it was also able to oxidize several common peroxidase substrates, indicating a broad range of substrate specificity that is characteristic of class III plant peroxidases. Cytochemical studies showed that the enzyme is localized in C. roseus mesophyll vacuoles, in individual spots at the inner surface of the tonoplast. This particular location suggests a meaningful spatial organization that led to the proposal of a metabolic channeling model for the peroxidase-mediated synthesis of AVLB. The importance of this type of mechanism in the regulation of peroxidase isoenzyme functions in vivo is discussed. In view of the results obtained it is concluded that the basic peroxidase present in C. roseus leaves fulfills all the requirements to be considered as an AVLB synthase, and it is proposed that this specific function of this multifunctional enzyme is determined by metabolic channeling resulting from specific protein-protein interactions.

Staining of xylem parenchyma mitochondria with photo-oxidized 3,3'-diaminobenzidine.

A photo-oxidized solution of 3,3'-diaminobenzidine (DAB) is used to stain xylem parenchyma mitochondria in specimens prepared from lupin hypocotyls fixed with glutaraldehyde and osmium tetroxide and embedded in Epon. No other subcellular components, including plastids, nuclei, vacuoles or cell walls were stained when xylem parenchyma cells were exposed to this reagent for 1 hr. This reaction was stable for 20 min at 80 C, inhibited by KCN, and insensible to 3-amino-1,2,4-triazole. The outstanding sensitivity of this reaction to inhibition probes suggests that this stain is analogous to the previously described DAB/cytochrome c/cytochrome oxidase reaction in plant mitochondria, although the incubation of lupin sections with freshly prepared DAB solution (free of auto-oxidized DAB) did not result in staining. These results draw attention to the unreliability of DAB oxidation for demonstrating electron transport in plant mitochondria. However, we do recommend photo-oxidized DAB as a direct ultrastructural stain for plant mitochondria without reference to its oxidative capacity.

Zinnia elegans uses the same peroxidase isoenzyme complement for cell wall lignification in both single-cell tracheary elements and xylem vessels.

The nature of the peroxidase isoenzyme complement responsible for cell wall lignification in both Zinnia elegans seedlings and Z. elegans tracheary single-cell cultures have been studied. Results showed that both hypocotyls and stems from lignifying Z. elegans seedlings express a cell wall-located basic peroxidase of pI approximately 10.2, which was purified to homogeneity. Molecular mass determination under non-denaturing conditions showed an M(r) of about 43 000, similar to that of other plant peroxidases. The purified Z. elegans peroxidase showed absorption maxima at 403 (Soret band), and at 496-501 and 632-635 (alpha and beta absorption bands), indicating that this enzyme is a high spin ferric haem protein, belonging to the plant peroxidase superfamily, the prosthetic group being ferric protoporphyrin IX. The N-terminal amino acid sequence of this Z. elegans basic peroxidase was KVAVSPLS (peptide motif in bold), which shows strong homologies with the N-amino acid terminus of other strongly basic plant peroxidases. Isoenzyme and western blot analyses showed that this peroxidase isoenzyme is also expressed in trans-differentiating Z. elegans tracheary single-cell cultures. The results also showed that Z. elegans tracheary single-cell cultures not only express the same peroxidase isoenzyme as the Z. elegans lignifying xylem, but that this peroxidase isoenzyme acts as a marker of tracheary element differentiation in Z. elegans mesophyll single-cell cultures. From these results, it may be concluded that Z. elegans uses a single programme, i.e. an identical peroxidase isoenzyme complement, for lignification of the xylem, regardless of the existence of different ontogenesis pathways from either mesophyll cells (in the case of tracheary elements) or cambial derivatives (in the case of xylem vessels).

Hammett rho sigma correlation for the inhibition by indoles of coniferyl alcohol oxidation catalyzed by cell wall peroxidases.

The inhibitory effect of indole-3-acetic acid, and of its peroxidase-mediated degradation products of an indole nature, on the oxidation rate of coniferyl alcohol catalyzed by cell wall peroxidases has been studied. The results show that the inhibitory effect of indole-3-acetic acid and indole-3-carbinol may be explained, in part, by their properties as peroxidase substrates. However, I50 values for a series of indole compounds not regarded as peroxidase substrates show a good correlation with the electron-donating or electron-withdrawing nature of the 3-substituents, as judged by the linearity of the Hammett rho sigma plot. These results suggest that although the properties of indole compounds as peroxidase substrates may be responsible, in part, for their inhibitory effects on the peroxidase-mediated oxidation of coniferyl alcohol, the inhibitory effect appears to be mainly determined by the acidity of the imino group of the indole nucleus.

Zymographic screening of plant peroxidase isoenzymes oxidizing 4-hydroxystilbenes.

A zymographic assay is described for the detection of peroxidase isoenzymes oxidizing 4-hydroxystilbene following isoelectric focusing. The assay is based on coupling intermediate products of the oxidation of 4-hydroxystilbene with 4-aminoantipyrine, with resultant formation of dye complexes. Control experiments in the absence of 4-hydroxystilbene and hydrogen peroxide demonstrate the peroxidative nature of the 4-hydroxystilbene-dependent dye reaction.

The spectrophotometric determination of indole-3-methanol in small samples with p-dimethylaminocinnamaldehyde on the basis of the formation of an azafulvenium salt.

Indole-3-methanol is coupled, at acidic pH, with p-dimethylaminocinnamaldehyde to give a highly colored azafulvenium salt. IR, 1H NMR, and mass spectroscopic evidence indicates that this azafulvenium salt is the 2-[3'-(p-dimethylaminophenyl)-2'-propenyliden]-3-hydroxymethyl-2H- indolenine hydrochloride. This reaction led us to elaborate a rapid colorimetric assay for quantitative determination of indole-3-methanol formed by peroxidases as the product of oxidation of the plant growth regulator indole-3-acetic acid.

Indole-3-methanol is the main product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases from Lupinus.

The nature of the products of the auxin catabolism mediated by both basic and acidic isoperoxidases has been studied. While indole-3-methanol is only a minor product of the oxidation of indole-3-acetic acid catalyzed by extracellular acidic isoperoxidases, it is the only product of the oxidation of indole-3-acetic acid catalyzed by two cytosolic basic isoperoxidases (EC 1.11.1.7) from lupin (Lupinus albus L.) hypocotyls. The putative indole-3-methanol formed by these latter isoperoxidases was isolated and then characterized by mass spectrometry and (1)H-nuclear magnetic resonance spectrometry. These results are discussed with respect to the diversity and compartmentation of the catabolism of indole-3-acetic acid in plant tissues.