A. Ros Barceló

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.

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

Abstract. The nature of the enzymatic system responsible for the generation of H2O2 in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H2O2 production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H2O2 and to sustain H2O2 production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H2O2 production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H2O2 production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H2O2 production in lignifying xylem was obtained from the observation that areas of H2O2 production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H2O2 which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H2O2-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H2O2 to peroxidase in the lignifying xylem of Z. elegans.The nature of the enzymatic system responsible for the generation of H2O2 in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H2O2 production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H2O2 and to sustain H2O2 production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H2O2 production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H2O2 production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H2O2 production in lignifying xylem was obtained from the observation that areas of H2O2 production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H2O2 which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H2O2-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H2O2 to peroxidase in the lignifying xylem of Z. elegans.

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

Summary. 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 (α,β-unsaturated) cinnamaldehyde functional group, which is responsible for the dye reaction, is lost due to lateral chain cross-linking reactions involving the β carbon. Fourier transform infrared spectroscopy and thioacidolysis analyses of phloroglucinol-positive lignifying plant cell walls belonging to the plant species Zinnia elegans L., Capsicum annuum var. annuum, Populus alba L., and Pinus halepensis L. 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.

Purification and characterization of α‐3′,4′‐anhydrovinblastine synthase (peroxidase‐like) from Catharanthus roseus (L.) G. Don

An H2O2‐dependent enzyme capable of coupling catharanthine and vindoline into α‐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.

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 Mr (determined by matrix-assisted laser-desorption ionization time of flight) of 34,700 (ZePrx34.70) and a Mr 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. Mr 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 kcat and apparent KmRH 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.

Effect of dimethyl-β-cyclodextrins on resveratrol metabolism in Gamay grapevine cell cultures before and after inoculation with shape Xylophilus ampelinus

Gamay cell cultures were treated with dimethyl-β-cyclodextrins in order to ascertain their effect on resveratrol metabolism before and after inoculation with Xylophilus ampelinus. The results showed that, in grapevine cell suspensions, dimethyl-β-cyclodextrins themselves do not need the co-cultivation with bacteria to act as elicitors of the cells producing trans-resveratrol, a phytoalexin of grapevines. Dimethyl-β-cyclodextrins protected cell suspensions against bacteria by maintaining a high level of peroxidase activity.

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.

Peroxidase and not laccase is the enzyme responsible for cell wall lignification in the secondary thickening of xylem vessels inLupinus

SummaryThe post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3′-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H2O2 and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3′-diaminobenzidine both in the presence and in the absence of H2O2. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3′-diaminobenzidine and coniferyl alcohol in the presence of H2O2. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.The post-exponential growth phase of lupin (Lupinus albus cv. Multolupa) hypocotyls is characterized by a strong deposition of lignins in the primary and secondary walls of the xylem vessels. Coinciding with this phenomenon, there is a clearly peroxidatic activity in both the primary cell walls and the outer-most layers of the secondary thickening of the xylem vessels, as demonstrated by 3,3′-diaminobenzidine cytochemistry. This activity was completely inhibited by KCN and the removal of H2O2 and was not due to laccase since this enzyme shows an almost total inability to oxidize 3,3′-diaminobenzidine both in the presence and in the absence of H2O2. The absence of laccase-like activities in cell walls of vascular cells was supported by the fact that cell wall proteins from vascular cells were only capable of oxidizing 3,3′-diaminobenzidine and coniferyl alcohol in the presence of H2O2. These results support the idea of an exclusive role of peroxidase (and exclude any role for laccase) in lignin formation in the secondary thickening of xylem vessels inLupinus.

A basic peroxidase isoenzyme from vacuoles and cell walls of Vitis vinifera.

Abstract The substrate profile of a basic peroxidase isoenzyme located in vacuoles and cell walls from Vitis vinifera and efficacy in oxidizing both vacuolar and cell wall phenolic substrates was studied. The reactivity of this isoenzyme with H 2 O 2 [ k 1 (CoI formation constant) = 1.1 (quercetin), 1.6 (myricetin), 1.7 ( trans -resveratrol) and 10.4 (coniferyl alcohol) μM −1 sec −1 ] and with the phenolics k 3 (CoII reduction constant) =20.7 (quercetin), 6.9 (myricetin), 11.9 ( trans -resveratrol) and 8.7 (coniferyl alcohol) μM −1 sec −1 ], suggests that the isoenzyme reacts with H 2 O 2 with a similar reactivity to that shown by other peroxidases, and that both vacuolar and cell wall phenolics are excellent substrates for CoII reduction.