Oxidation of cinnamyl alcohols and aldehydes by a basic peroxidase from lignifying Zinnia elegans hypocotyls.

The xylem of 26-day old Zinnia elegans hypocotyls synthesizes lignins derived from coniferyl alcohol and sinapyl alcohol with a G/S ratio of 43/57 in the aryl-glycerol-beta-aryl ether core, as revealed by thioacidolysis. Thioacidolysis of Z. elegans lignins also reveals the presence of coniferyl aldehyde end groups linked by beta-0-4 bonds. Both coniferyl and sinapyl alcohols, as well as coniferyl and sinapyl aldehyde, are substrates of a xylem cell wall-located strongly basic peroxidase, which is capable of oxidizing them in the absence and in the presence of hydrogen peroxide. This peroxidase shows a particular affinity for cinnamyl aldehydes with kappa(M) values in the mu(M) range, and some specificity for syringyl-type phenols. The affinity of this strongly basic peroxidase for cinnamyl alcohols and aldehydes is similar to that shown by the preceding enzymes in the lignin biosynthetic pathway (microsomal 5-hydroxylases and cinnamyl alcohol dehydrogenase), which also use cinnamyl alcohols and aldehydes as substrates, indicating that the one-way highway of construction of the lignin macromolecule has no metabolic "potholes" in which the lignin building blocks might accumulate. This fact suggests a high degree of metabolic plasticity for this basic peroxidase, which has been widely conserved during the evolution of vascular plants, making it one of the driving forces in the evolution of plant lignin heterogeneity.     

Effect of copper excess on H2O2 accumulation and peroxidase activities in bean roots

We studied oxidative stress and peroxidase activity resulting from application of excess copper in the nutrient medium on the roots of young bean seedlings. The change in H2O2 content, lipid peroxidation and antioxidant enzymes activities were quantified and located. Excess of copper caused a loss of membrane integrity and the formation of hydrogen peroxide (H2O2) as visualized in the transmission electron microscopy and measured using spectrophotometry. H2O2 accumulated in the intercellular spaces and in the cell wall. The production of H2O2 was accompanied by an increase in the activity of soluble and ionic GPX (guaiacol peroxidase, EC 1.11.17), CAPX (coniferyl alcohol peroxidase) and NADH oxidase.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

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.     

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).     

Differential effects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans

IWF, intercellular washing fluid
pCMB, p-chloromercuribenzoic acid
SNAP, S-nitroso-N-acetyl-penicillamine SNP, sodium nitroprusside
TMB, 3,3’,5,5’- tetramethylbenzidine

Sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) are two nitric oxide (NO)-releasing compounds that, when used at 5·0 mol m^–3 concentrations, are capable of releasing NO in the aqueous phase at a rate of 35 ± 4 and 47 ± 5 μmol m^–3 s^–1, respectively. For this reason, the effect of SNP and SNAP on coniferyl alcohol peroxidase and on H₂O₂ production by the lignifying xylem of Zinnia elegans (L.) has been studied in order to ascertain whether NO, which is a synchronizing chemical messenger in animals and an air pollutant, has any effect on these plant-specific metabolic aspects. The results showed that both SNP and SNAP provoke an inhibition in the mol m^–3 concentration range of the coniferyl alcohol peroxidase activity of a basic peroxidase isoenzyme present in the intercellular washing fluid of Z. elegans. The effect of these NO-releasing compounds on peroxidase was confirmed through histochemical studies, which showed that xylem peroxidase was totally inhibited by treatment with these NO donors at 5·0 mol m^–3, and by NO at a concentration change rate of 55 ± 5 and 110 ± 9 μmol m^–3 s^–1. However, SNP, at 5·0 mol m^–3, does not have any effect on H₂O₂ production by the xylem of Z. elegans. The fact that SNP and SNAP are two structurally dissimilar compounds which only share the common ability to release NO in aqueous buffer, and that similar results were obtained when using NO itself, suggest that NO could be considered as an inhibitor of coniferyl alcohol peroxidase which does not affect H₂O₂ production in the xylem of Z. elegans.     

On the mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 by EDTA.

The mechanism of inhibition of the veratryl alcohol oxidase activity of lignin peroxidase H2 (LiPH2) by EDTA was investigated. It was found that EDTA was decarboxylated and that cytochrome c, nitro blue tetrazolium, ferric iron, and molecular oxygen were reduced in a reaction mixture containing LiPH2, H2O2, veratryl alcohol, and EDTA. The reductive activity observed with LiPH2 followed first order kinetics with respect to the concentration of EDTA. Stoichiometry studies showed that in the presence of sufficient EDTA, 1.7 mol of ferric iron were reduced per mole of H2O2 added to the reaction mixture. Superoxide- and EDTA-derived radicals were detected by ESR spin trapping upon incubation of LiPH2 with H2O2, veratryl alcohol, and EDTA. The Km values of veratryl alcohol and H2O2 remained the same for both the oxidative and reductive activities of LiPH2. Reductive activity was also observed with LiPH2 and EDTA using other free radical mediators in the place of veratryl alcohol, such as 1,4-dimethoxybenzene, 1,2,3- and 1,2,4-trimethoxybenzenes, and 1,2,4,5-tetramethoxybenzene. EDTA reduced the cation radical of 1,2,4,5-tetramethoxybenzene formed by LiPH2 in the presence of H2O2. Hence, it is proposed that the apparent inhibition of the veratryl alcohol oxidase activity of LiPH2 by EDTA is due to the reduction of the veratryl alcohol cation radical intermediate back to veratryl alcohol by EDTA. The reduction of cytochrome c, nitro blue tetrazolium, ferric ion, and molecular oxygen appears to be mediated by the EDTA radical formed by reduction of the veratryl alcohol cation radical.     

Apoplastic Peroxidases and Lignification in Needles of Norway Spruce (Picea abies L.)

The objective of the present study was to investigate the correlation of soluble apoplastic peroxidase activity with lignification in needles of field-grown Norway spruce (Picea abies L.) trees. Apoplastic peroxidases (EC 1.11.1.7) were obtained by vacuum infiltration of needles. The lignin content of isolated cell walls was determined by the acetyl bromide method. Accumulation of lignin and seasonal variations of apoplastic peroxidase activities were studied in the first year of needle development. The major phase of lignification started after bud break and was terminated about 4 weeks later. This phase correlated with a transient increase in apoplastic guaiacol and coniferyl alcohol peroxidase activity. NADH oxidase activity, which is thought to sustain peroxidase activity by production of H2O2, peaked sharply after bud break and decreased during the lignification period. Histochemical localization of peroxidase with guaiacol indicated that high activities were present in lignifying cell walls. In mature needles, lignin was localized in walls of most needle tissues including mesophyll cells, and corresponded to 80 to 130 [mu]mol lignin monomers/g needle dry weight. Isoelectric focusing of apoplastic washing fluids and activity staining with guaiacol showed the presence of strongly alkaline peroxidases (isoelectric point [greater than or equal to] 9) in all developmental stages investigated. New isozymes with isoelectric points of 7.1 and 8.1 appeared during the major phase of lignification. These isozymes disappeared after lignification was terminated. A strong increase in peroxidase activity in autumn was associated with the appearance of acidic peroxidases (isoelectric point [less than or equal to] 3). These results suggest that soluble alkaline apoplastic peroxidases participate in lignin formation. Soluble acidic apoplastic peroxidases were apparently unrelated to developmentally regulated lignification in spruce needles.     

Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

 Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl₂ extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees. Received: 20 May 1997 / Accepted: 7 August 1997     

An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis     

Phenol removal using Brassica juncea hairy roots: role of inherent peroxidase and H(2)O(2)

Removal of phenol, a major pollutant in aqueous effluents was studied using plant hairy root cultures. Among four different species of hairy roots tested, Brassica juncea showed the highest potential for phenol removal. The effect of phenol concentration and reuse in a batch system was studied using B. juncea hairy root cultures. Unlike most of the studies reported earlier, phenol removal by the hairy roots was seen to take place without the need for addition of external hydrogen peroxide (H(2)O(2)). To understand the mechanism of phenol removal, levels of peroxidase and phenol oxidase were monitored in the hairy roots. Peroxidase activity in the roots was enhanced when exposed to phenol, while phenol oxidase remained constant. Since peroxidase has a pre-requisite for H(2)O(2), the levels of H(2)O(2) were monitored for its in situ synthesis. H(2)O(2) levels were seen to increase in the presence of phenol. Thus, a mechanism wherein hairy roots also produce H(2)O(2) besides peroxidase, as a protection strategy of plant against xenobiotic stress is plausible.     

Cell-wall proteins from Sitka spruce xylem are selectively insolubilised during formation of dehydrogenation polymers of coniferyl alcohol

Extracts from the lignifying xylem of Sitka spruce that were enriched in cell-wall-associated glycoproteins contained peroxidase and oxidase activity and readily formed lignin-like water-insoluble dehydrogenation polymers (DHPs) from coniferyl alcohol (CA) when supplied with H2O2. During the formation of DHPs, the abundance of a number of polypeptides in the extracts was diminished. However, these polypeptides were also diminished in control reactions that contained H2O2 but lacked CA. Polypeptides could be recovered from the DHPs by heating in SDS-PAGE sample buffer but no insolubilised polypeptides could be recovered from the + H2O2 reactions. Although most of the DHP-bound polypeptides were easily removed by pre-washing the DHPs, two polypeptides at 125 and 52 kDa remained tightly bound to the DHPs. The abundance of the two DHP-bound polypeptides mirrored the diminution of 120 and 46 kDa polypeptides in the extracts. The N-terminal protein sequences of the 125 and 52 kDa DHP-bound polypeptides were essentially identical to the sequences obtained from the 120 and 46 kDa polypeptides from the extracts, which confirmed that the DHP-bound polypeptides were derived from these soluble polypeptides. The 125-kDa DHP-bound polypeptide yielded an N-terminal protein sequence that was identical to a laccase-type oxidase previously identified in similar extracts from lignifying Sitka xylem. The N-terminal protein sequence of the 46-kDa polypeptide was homologous with a subset of plant peroxidases. The DHPs had tightly bound peroxidase and oxidase activity, which suggested that these polypeptides were active in their insolubilised state. The mechanism and selectivity of insolubilisation of these enzymes is discussed.     

Perturbed heme binding is responsible for the blistering phenotype associated with mutations in the Caenorhabditis elegans dual oxidase 1 (DUOX1) peroxidase domain

Dual oxidase (DUOX) enzymes support a wide variety of essential reactions, from cellular signaling to thyroid hormone biosynthesis. In Caenorhabditis elegans, the DUOX system (CeDUOX1/2) plays a crucial role in innate immunity and in stabilizing the cuticle by forming tyrosine cross-links. The current model suggests that superoxide generated by CeDUOX1 at the C-terminal NADPH oxidase domain is rapidly converted to H(2)O(2). The H(2)O(2) is then utilized by the N-terminal peroxidase-like domain to cross-link tyrosines. We have now created a series of mutations in the isolated peroxidase domain, CeDUOX1(1-589). One set of mutations investigate the roles of a putative distal tyrosine (Tyr(105)) and Glu(238), a proposed covalent heme-binding residue. The results confirm that Glu(238) covalently binds to the heme group. A second set of mutations (G246D and D392N) responsible for a C. elegans blistering cuticle phenotype was also investigated. Surprisingly, although not among the catalytic residues, both mutations affected heme co-factor binding. The G246D mutant bound less total heme than the wild type, but a higher fraction of it was covalently bound. In contrast, the D392N mutant appears to fold normally but does not bind heme. Molecular dynamics simulations of a CeDUOX1(1-589) homology model implicate displacements of the proximal histidine residue as the likely cause. Both enzymes are structurally stable and through altered heme interactions exhibit partial or complete loss of tyrosine cross-linking activity, explaining the blistering phenotype. This result argues that the CeDUOX peroxidase domain is primarily responsible for tyrosine cross-linking.     

NAD(P)H oxidase and peroxidase activities in purified plasma membranes from cauliflower inflorescences

An NAD(P)H oxidase activity stimulated by phenolic compounds has been investigated in purified plasma membranes (pm) and in an intracellular membrane (icm) fraction depleted in plasma membranes, both obtained from a microsomal fraction from cauliflower inflorescences ( Brassica oleracea L.). The phenolic compounds salicylhydroxamic acid (SHAM), ferulic acid, coniferyl alcohol, n -propyl gallate, naringenin, kaempferol and caffeic acid all strongly stimulated the activity. Peroxidase (EC 1.11.1.7), or a peroxidase-like enzyme, was responsible for the NAD(P)H oxidase activity, which proceeded through a free-radical chain reaction and was inhibited by catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and KCN. Most of the total activity was soluble; however, the membrane-bound activity was highly enriched in the pm compared to the icm. The catalase activity was 6 times higher in the icm-fraction than in the pm-fraction, but this was not the reason for the much lower phenol-stimulated NADH oxidase activity in the icm. Peroxidase activity measured with o -dianisidine and H₂O₂ had about the same specific activities in the pm-and icm-fractions.
Neither the phenol-stimulated NADH oxidase nor the peroxidase activity could be washed away from the pm even by 0.7 M NaCl, indicating that these activities are truly membrane-bound. SHAM as well as the other phenolic compounds capable of stimulating the NADH oxidase reaction were potent inhibitors of blue light-induced cytochrome b -reduction in the pm fraction.     

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis)

Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell wall but it could be extracted by elution with salt solutions such as 1 M NaCl or CaCl₂. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent M_r approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent M_r approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases (EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed. Received: 9 January 1997 / Accepted: 14 March 1997     

Lignin and veratryl alcohol are not inducers of the ligninolytic system of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. In this work, we investigated the roles of veratryl alcohol and lignin in the ligninolytic system of P. chrysosporium BKM-F-1767 cultures grown under nitrogen-limited conditions. Cultures supplemented with 0.4 to 2 mM veratryl alcohol showed increased lignin peroxidase activity. Addition of veratryl alcohol had no effect on Mn-dependent peroxidase activity and inhibited glyoxal oxidase activity. Azure-casein analysis of acidic proteases in the extracellular fluid showed that protease activity decreased during the early stages of secondary metabolism while lignin peroxidase activity was at its peak, suggesting that proteolysis was not involved in the regulation of lignin peroxidase activity during early secondary metabolism. In cultures supplemented with lignin or veratryl alcohol, no induction of mRNA coding for lignin peroxidase H2 or H8 was observed. Veratryl alcohol protected lignin peroxidase isozymes H2 and H8 from inactivation by H2O2. We conclude that veratryl alcohol acts as a stabilizer of lignin peroxidase activity and not as an inducer of lignin peroxidase synthesis.     

beta-fluoro-coniferyl alcohol does not inhibit lignin biosynthesis in suspension cultures of Picea abies (L.) Karst

A fluorinated analogue of coniferyl alcohol has been reported to be a specific inhibitor of oxidases involved in the biosynthesis of lignin. The Z isomer of beta-fluoro-coniferyl alcohol was synthesized and used for the preparation of dehydrogenation polymers (DHPs) and was also tested on lignin producing suspension cultures of spruce (Picea abies (L.) Karst.). The growth of the cells or the production of lignin by the suspension cultures was not significantly affected by the addition of fluoroconiferyl alcohol. This analogue did not form polymers quite as easily as did coniferyl alcohol in oxidation with hydrogen peroxide and horseradish peroxidase. In both cases the beta-fluoroconiferyl alcohol became incorporated in the polymeric product. We were unable to detect any specific inhibition of peroxidase activity, which is at variance with earlier reports of pronounced inhibition of lignin biosynthesis in poplar plantlets by fluoroconiferin, a potential inhibitor of oxidases involved in lignin biosynthesis.     

Reduction of phenoxyl radicals mediated by monodehydroascorbate reductase

Monodehydroascorbate (MDA) reductase catalyzes the reduction of MDA, the only organic radical substrate for the enzyme reported so far. Here, we show that cucumber MDA reductase is also capable of reducing phenoxyl radicals which are generated by horseradish peroxidase (HRP) with H2O2. The addition of MDA reductase plus NADH suppressed the HRP/H2O2 dependent oxidation of quercetin, accompanied by the oxidation of NADH. The quenching of the quercetin radical by MDA reductase plus NADH was confirmed by ESR. MDA reductase with NADH also suppressed the HRP/H2O2 dependent oxidation of hydroxycinnamates, including ferulic acid, coniferyl alcohol, and chlorogenic acid. Thus, the phenoxyl radicals of plant phenols can be reduced to their respective parent phenols by MDA reductase via a mechanism similar to the reduction of MDA.     

Ascorbic acid and flavonoid-peroxidase reaction as a detoxifying system of H(2)O(2) in grapevine leaves

Biosynthesis of both ascorbic acid (AsA) and peroxidase activity were induced by light in cv. Sultana grapevine leaves. Induced peroxidase activity mainly involved basic isoenzymes of pI 9.8 and 9.6 and catalyzed the oxidation of flavonoids like quercetin and kaempferol and derivatives of hydroxycinnamic acids such as ferulic and p-coumaric acids, but not AsA. However, the peroxidase-dependent oxidation of ferulic acid and quercetin was temporarily suppressed by AsA as long as it remained in the reaction medium. Kinetics and spectroscopic results indicated that AsA was oxidized to dehydroascorbic acid only in the presence of phenols or flavonoids, and did not interfere with the catalytic activity of the peroxidase. Ascorbate peroxidase isoenzymes (APx), whose activities are widely considered central for detoxification of H(2)O(2) in most plant cells, were not detected in grape leaves extracts. The significance of light stimulus on peroxidase activity and leaf AsA content is discussed in terms of a flavonoid-redox cycle proposed as an alternative system to detoxify H(2)O(2) in grapevine leaves.