Apoplastic peroxidases and ascorbate are involved in manganese toxicity and tolerance of Vigna unguiculata

Excessive manganese (Mn) supply induced the formation of brown spots on leaves as typical Mn toxicity symptoms in cowpea ( Vigna unguiculata L. Walp.) grown in hydroponics. Differences in Mn resistance between cv. TVu 91 (Mn-sensitive) and cv. TVu 1987 (Mn-tolerant) expressed in the density of brown spots in older leaves were due to higher Mn tissue tolerance. Apoplastic water-soluble peroxidase (POD) in the apoplastic washing fluid (AWF) was enhanced by increasing Mn leaf content and generally significantly higher in leaves of cv. TVu 91 than in cv. TVu 1987. Electrophoresis of AWF revealed the presence of several water-soluble POD isoenzymes. At toxic Mn supply, the activities of these and additional POD isoenzymes increased more in the Mn-sensitive cultivar. Levels of ascorbic acid in the apoplast and cytoplasm of the Mn-sensitive cv. TVu 91 decreased with increasing leaf Mn contents, whereas Mn-tolerant cv. TVu 1987 was not affected. Mn treatment lead to a stimulation of the enzymes of the ascorbic acid regeneration system (monodehydroascorbic acid reductase and glutathione reductase) in both cultivars, but the activation of glutathione reductase was clearly more enhanced in the Mn-tolerant cultivar TVu 1987. The results provide circumstantial evidence that apoplastic ascorbate and peroxidases are involved in the expression of Mn toxicity and genotypic Mn tolerance.     

The role of hydrogen peroxide-producing and hydrogen peroxide-consuming peroxidases in the leaf apoplast of cowpea in manganese tolerance

The apoplast is considered the leaf compartment decisive for manganese (Mn) toxicity and tolerance in cowpea (Vigna unguiculata). Particularly apoplastic peroxidases (PODs) were proposed to be key enzymes in Mn toxicity-induced processes. The presented work focuses on the characterization of the role of hydrogen peroxide (H2O2)-producing (NADH peroxidase) and H2O2-consuming peroxidase (guaiacol POD) in the apoplastic washing fluid (AWF) of leaves for early stages of Mn toxicity and genotypic differences in Mn tolerance of cowpea. Leaf AWF of the Mn-sensitive cultivar (cv) TVu 91 but not of the Mn-tolerant cv 1987 showed an increase of guaiacol-POD and NADH-peroxidase activities at elevated AWF Mn concentrations. two-dimensional resolutions of AWF proteins revealed that cv TVu 91 expressed more and additional proteins at high Mn treatment, whereas Mn-tolerant cv TVu 1987 remained nearly unaffected. In both cultivars, NADH-peroxidase activity and accompanied H2O2 formation rate in vitro were significantly affected by Mn2+, p-coumaric acid, and metabolites occurring in the AWF. The total phenol concentration in the AWF was indicative of advanced stages of Mn toxicity but was rather unrelated to early stages of Mn toxicity and genotypic differences in Mn tolerance. The NADH oxidation by AWF PODs was significantly delayed or enhanced in the presence of the protein-free AWF from cv TVu 1987 or cv TVu 91, respectively. High-performance liquid chromatography analysis of AWF indicates the presence of phenols in cv TVu 1987 not observed in cv TVu 91. We conclude from our studies that the H2O2-producing NADH peroxidase and its modulation by stimulating or inhibiting phenolic compounds in the leaf apoplast play a major role for Mn toxicity and Mn tolerance in cowpea.     

Light and Excess Manganese : Implications for Oxidative Stress in Common Bean

The effect of light intensity on antioxidants, antioxidant enzymes, and chlorophyll content was studied in common bean (Phaseolus vulgaris L.) exposed to excess Mn. Leaves of bean genotypes contrasting in Mn tolerance were exposed to two different light intensities and to excess Mn; light was controlled by shading a leaflet with filter paper. After 5 d of Mn treatment ascorbate was depleted by 45% in leaves of the Mn-sensitive genotype ZPV-292 and by 20% in the Mn-tolerant genotype CALIMA. Nonprotein sulfhydryl groups and glutathione reductase were not affected by Mn or light treatment. Ten days of Mn-toxicity stress increased leaf ascorbate peroxidase activity of cv ZPV-292 by 78% in low light and by 235% in high light, and superoxide dismutase activity followed a similar trend. Increases of ascorbate peroxidase and superoxide dismutase activity observed in cv CALIMA were lower than those observed in the susceptible cv ZPV-292. The cv CALIMA had less ascorbate oxidation under excess Mn-toxicity stress. Depletion of ascorbate occurred before the onset of chlorosis in Mn-stressed plants, especially in cv ZPV-292. Lipid peroxidation was not detected in floating leaf discs of mature leaves exposed to excess Mn. Our results suggest that Mn toxicity may be mediated by oxidative stress, and that the tolerant genotype may maintain higher ascorbate levels under stress than the sensitive genotype.     

Changes in Peroxidase Activity Associated with Cell Walls During Pine Hypocotyl Growth

Peroxidase active against 2,2'-azino-bis-[3-ethylbenzthiazoline-6-sulphonicacid] (ABTS) and guaiacol were found in the apoplastic fluid,as well as ionically and covalently associated with pine cellwalls. The highest activity was found covalently bound to cellwalls, while the lowest activity was in the apoplastic fluid.Both ABTS and guaiacol peroxidases increased with the hypocotylage in the three fractions, apoplastic, ionically and covalentlybound. Furthermore, the changes in both peroxidases along thehypocotyl were also studied. Both apoplastic ABTS- and guaiacol-peroxidasesincreased from the apical towards the basal region of the hypocotylsof 10-d-old seedlings. A relation between peroxidase activityin the apoplastic fluid and the cell wall stiffening in pinehypocotyls is proposed.Copyright 1995, 1999 Academic Press Cell wall, growth, hypocotyl, peroxidase, pine, Pinus pinaster Aiton     

Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.     

Localization and Characterization of Peroxidases in the Mitochondria of Chilling-Acclimated Maize Seedlings

We present evidence of two peroxidases in maize (Zea mays L.) mitochondria. One of these uses guaiacol and the other uses cytochrome c as the electron donor. Treatments of fresh mitochondria with protease(s) indicate that ascorbate and glutathione peroxidases are likely bound to the mitochondria as cytosolic contaminants, whereas guaiacol and cytochrome peroxidases are localized within the mitochondria. These two mitochondrial peroxidases are distinct from contaminant peroxidases and mitochondrial electron transport enzymes. Cytochrome peroxidase is present within the mitochondrial membranes, whereas guaiacol peroxidase is loosely bound to the mitochondrial envelope. Unlike other cellular guaiacol peroxidases, mitochondrial guaiacol peroxidase is not glycosylated. Digestion of lysed mitochondria with trypsin activated mitochondrial guaiacol peroxidase but inhibited cytochrome peroxidase. Isoelectric focusing gel analysis indicated guaiacol peroxidase as a major isozyme (isoelectric point 6.8) that is also activated by trypsin. No change in the mobility of guaiacol peroxidase due to trypsin treatment on native polyacrylamide gel electrophoresis was observed. Although both peroxidases are induced by chilling acclimation treatments (14[deg]C), only cytochrome peroxidase is also induced by chilling (4[deg]C). Because chilling induces oxidative stress in the maize seedlings and the mitochondria are a target for oxidative stress injury, we suggest that mitochondrial peroxidases play a role similar to catalase in protecting mitochondria from oxidative damage.     

The Amino Acid Sequence of Ascorbate Peroxidase from Tea Has a High Degree of Homology to That of Cytochrome c Peroxidase from Yeast

The chloroplast isozyme of ascorbate peroxidase from tea leaveswas digested with lysyl endopeptidase, and the amino acid sequencesof the peptide fragments were determined. These sequences accountedfor 64% of the amino acids in the entire protein. The sequenceof one of the peptides can be aligned with the region whichincludes the proximal histidine that serves as the fifth ligandof the heme iron in guaiacol peroxidases and cytochrome c peroxidase.The sequences of the peptides from ascorbate peroxidase exhibita higher degree of homology to the sequence of cytochrome cperoxidase from yeast than to those of guaiacol peroxidasesfrom plants. In addition, three of the peptides from ascorbateperoxidase show a high degree of homology to triose-phosphateisomerase from maize. From the available amino acid sequencesand the enzymatic and molecular properties of ascorbate andcytochrome c peroxidases, we propose that these hydrogen peroxide-scavengingperoxidases that use either cytochrome c or ascorbate as theelectron donor originated from the same ancestral protein. (Received July 5, 1991; Accepted December 6, 1991)     

Physiological and proteomic characterization of manganese sensitivity and tolerance in rice (Oryza sativa) in comparison with barley (Hordeum vulgare)

Background and Aims

Research on manganese (Mn) toxicity and tolerance indicates that Mn toxicity develops apoplastically through increased peroxidase activities mediated by phenolics and Mn, and Mn tolerance could be conferred by sequestration of Mn in inert cell compartments. This comparative study focuses on Mn-sensitive barley (Hordeum vulgare) and Mn-tolerant rice (Oryza sativa) as model organisms to unravel the mechanisms of Mn toxicity and/or tolerance in monocots.

Methods

Bulk leaf Mn concentrations as well as peroxidase activities and protein concentrations were analysed in apoplastic washing fluid (AWF) in both species. In rice, Mn distribution between leaf compartments and the leaf proteome using 2D isoelectic focusing IEF/SDS–PAGE and 2D Blue native BN/SDS–PAGE was studied.

Key Results

The Mn sensitivity of barley was confirmed since the formation of brown spots on older leaves was induced by low bulk leaf and AWF Mn concentrations and exhibited strongly enhanced H₂O₂-producing and consuming peroxidase activities. In contrast, by a factor of 50, higher Mn concentrations did not produce Mn toxicity symptoms on older leaves in rice. Peroxidase activities, lower by a factor of about 100 in the rice leaf AWF compared with barley, support the view of a central role for these peroxidases in the apoplastic expression of Mn toxicity. The high Mn tolerance of old rice leaves could be related to a high Mn binding capacity of the cell walls. Proteomic studies suggest that the lower Mn tolerance of young rice leaves could be related to Mn excess-induced displacement of Mg and Fe from essential metabolic functions.

Conclusions

The results provide evidence that Mn toxicity in barley involves apoplastic lesions mediated by peroxidases. The high Mn tolerance of old leaves of rice involves a high Mn binding capacity of the cell walls, whereas Mn toxicity in less Mn-tolerant young leaves is related to Mn-induced Mg and Fe deficiencies.     

Iron deficiency differently affects peroxidase isoforms in sunflower

The response of both specific (ascorbate peroxidase, APX) and unspecific (POD) peroxidases and H(2)O(2) content of sunflower plants (Helianthus annuus L. cv. Hor) grown hydroponically with (C) or without (-Fe) iron in the nutrient solution were analysed to verify whether iron deficiency led to cell oxidative status. In -Fe leaves a significant increase of H(2)O(2) content was detected, a result confirmed by electron microscopy analysis. As regards extracellular peroxidases, while APX activity significantly decreased, no change was observed in either soluble guaiacol or syringaldazine-dependent POD activity following iron starvation. Moreover, guaiacol-dependent POD activity was found to decrease in both ionically and covalently-cell-wall bound fractions, while syringaldazine-POD activity decreased only in the covalently-bound fraction. At the intracellular level both guaiacol-POD and APX activities underwent a significant decrease. The overall reduction of peroxidase activity was confirmed by the electrophoretic separation of POD isoforms and, at the extracellular level, by cytochemical localization of peroxidases by diaminobenzidine staining. The electrophoretic separation, besides quantitative differences, also revealed quantitative changes, particularly evident for ionically and covalently-bound fractions. Therefore, in sunflower plants, iron deficiency seems to affect the different peroxidase isoenzymes to different extents and to induce a secondary oxidative stress, as indicated by the increased levels of H(2)O(2). However, owing to the almost completely lack of catalytic iron capable of triggering the Fenton reaction, iron-deficient sunflower plants are probably still sufficiently protected against oxidative stress.     

Balance between anionic and cationic extracellular peroxidase activities in Sedum album leaves after ozone exposure. Analysis by high-performance liquid chromatography

Peroxidase activity was assayed with different electron donors (guaiacol, ascorbate, syringaldazine) in the intercellular fluid of Sedum album L. leaves after ozone exposure. Anionic and cationic peroxidases were separated and purified by high performance ion-exchange and gel permeation chromatography. Both isoperoxidases were tested as regards their molecular weight and apparent kinetic constants with different substrates. Ascorbate peroxidase activity was rapidly stimulated after ozone exposure, whereas syringaldazine peroxidase activity reached its maximum 24 h later. Increases in ascorbate and syringaldazine peroxidase activities occurred simultaneously with increases in cationic and anionic peroxidase activities, respectively. Apparent K_m values indicate a high affinity of cationic peroxidases for ascorbate and of anionic peroxidases for syringaldazine. The metabolic role of this balance between cationic and anionic peroxidases after ozone exposure is discussed.     

Surface chemical properties of purified root cell walls from two tobacco genotypes exhibiting different tolerance to manganese toxicity

Surface chemical characteristics of root cell walls extracted from two tobacco genotypes exhibiting differential tolerance to Mn toxicity were studied using potentiometric pH titration and Fourier transform infrared spectroscopy. The Mn-sensitive genotype KY 14 showed a stronger interaction of its cell wall surface with metal ions than did the Mn-tolerant genotype Tobacco Introduction (T.I.) 1112. This observation may be attributed to the relatively higher ratio of COO⁻ to COOH in KY 14 cell walls than that found in the cell walls of T.I. 1112 in the pH range of 4 to 10. For both genotypes, the strength of binding between metal ions and cell wall surface was in the order of Cu > Ca > Mn > Mg > Na. However, a slightly higher preference of Ca over Mn was observed with the T.I. 1112 cell wall. This may explain the high accumulation of Mn in the leaves of Mn-tolerant genotype T.I. 1112 rather than the high accumulation of Mn in roots, as occurred in Mn-sensitive KY 14. It is concluded that surface chemical characteristics of cell walls may play an important role in plant metal ion uptake and tolerance.     

Differential tolerance to Mn toxicity in perennial ryegrass genotypes: involvement of antioxidative enzymes and root exudation of carboxylates

Mechanisms underlying differential tolerance to Manganese (Mn) toxicity in perennial ryegrass (Lolium perenne L.) cultivars are poorly understood. We evaluated activity of antioxidative enzymes and root exudation of carboxylates in four ryegrass cultivars subjected to increasing Mn supply under nutrient solution conditions. A growth reduction caused by Mn toxicity was smaller in Jumbo and Kingston than Nui and Aries cultivars. Shoot Mn accumulation varied in the order Nui > Aries > Kingston > Jumbo. Ascorbate peroxidase and guaiacol peroxidase activities increased with Mn excess. Mn-tolerant Jumbo and Kingston had high activity of these enzymes and relatively low lipid peroxidation. Kingston was most tolerant to high tissue Mn concentrations and had the highest superoxide dismutase activity. Increased activity of antioxidative enzymes in Mn-tolerant cultivars could protect their tissues against oxidative stress triggered by Mn excess. Mn toxicity induced root exudation of carboxylates; oxalate and citrate may decrease Mn availability in the rhizosphere, thus enhancing Mn tolerance in ryegrass.     

The possible involvement of peroxidase in defense of yellow lupine embryo axes against Fusarium oxysporum

Peroxidase activity (EC 1.11.1.7) towards phenolic substrates, i.e. pyrogallol, syringaldazine and guaiacol, and ascorbate peroxidase activity (EC 1.11.1.11) were analyzed in embryo axes of Lupinus luteus L. cv. Polo cultured on Heller medium for 96h after inoculation with the necrotrophic fungus Fusarium oxysporum f.sp. Schlecht lupini. Four variants were compared: inoculated embryo axes cultured with 60mM sucrose (+Si) or without it (-Si), and non-inoculated embryo axes cultured with 60mM sucrose (+Sn) or without it (-Sn). Between 0 and 96h of culture, peroxidase activity towards the phenolic substrates increased in all variants except -Si, where a decrease was noted in peroxidase activity towards syringaldazine and guaiacol, but not towards pyrogallol. In +Si tissues, a considerable increase in enzyme activity towards these substrates was recorded starting from 72h of culture. Lignin content of +Si tissues increased already at the first stage of infection, i.e. 24h after inoculation. Additionally, in +Sn tissues, high ascorbate peroxidase activity was observed during the culture. Its activity increased in +Si tissues, beginning at 72h after inoculation. However, this was lower than in +Sn tissues. At 72h after inoculation, a considerably stronger development of the infection was observed in -Si than in +Si tissues during our earlier research [Morkunas, I. et al., 2005. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiol Biochem 2005; 43: 363-73]. Both peroxidases assayed towards phenolic substrates and ascorbate peroxidase was less active in -Si tissues than in -Sn tissues. Hydrogen peroxide concentration was much higher in -Si than in +Si tissues. These results indicate that peroxidases may be some of the elements of the defense system that are stimulated by sucrose in yellow lupine embryo axes in response to infection caused by F. oxysporum.     

Effects of cadmium and copper on peroxidase, NADH oxidase and IAA oxidase activities in cell wall, soluble and microsomal membrane fractions of pea roots

Twelve-day-old seedlings of pea (Pisum sativum L.) that were treated for 4 days by 20 and 100 micromol/l Cd(NO3)2 or CuSO4 showed a growth reduction in all organs. From root protein extracts, the activities of guaiacol peroxidase (GPX; EC 1.11.1.7), ascorbate peroxidase (APX; EC 1.11.1.11), coniferyl alcohol peroxidase (CAPX), NADH oxidase, and indole-3-acetic acid (IAA) oxidase were measured in covalently--and ionically--[symbol: see text] bound cell wall, soluble, and microsomal membrane fractions. With the exception of 20 micromol/l Cu, metal treatments enhanced GPX activity in all fractions. Only IAA oxidase activity was metal-elevated in the covalently bound cell wall fraction, while the ionic one showed Cd stimulation for all assayed enzymic activities. These effects were not entirely observed in Cu-treated plants, since APX and IAA oxidase activities were only enhanced in this fraction. However, soluble extract showed stimulation of APX activity, while in the microsomal fraction metal exposure also increased the activities of CAPX and NADH oxidase. Differential responses of root cell fractions to the presence of cadmium and copper ions are discussed in regard to the contribution of their enzymic capacities in antioxidant, lignification, and auxin degradation pathways. Comparisons between metals and dose effects are also underlined.     

Silicon ameliorates manganese toxicity by regulating manganese transport and antioxidant reactions in rice (Oryza sativa L.)

Background and aims

This study aimed to investigate the roles of silicon (Si) in ameliorating manganese (Mn) toxicity in two rice (Oryza sativa L.) cultivars: i.e. cv. Xinxiangyou 640 (XXY), a Mn-sensitive cultivar and cv. Zhuliangyou 99 (ZLY), a Mn-tolerant cultivar.

Methods

Plants were cultured in nutrient solution containing normal Mn (6.7 μM) or high Mn (2.0 mM), both with or without Si supply at 1.5 mM Si.

Results

Plant growth was severely inhibited by high Mn in cv. XXY, but was enhanced by Si supply. In cv. XXY, Si-enhanced tolerance resulted from a restriction of Mn transport, whereas in cv. ZLY Mn uptake was depressed. In cv. XXY, high Mn significantly increased superoxide dismutase (SOD), catalase and ascorbate peroxidase activities but decreased non-protein thiols and glutathione concentrations, leading to accumulation of H₂O₂ and malondialdehyde. The addition of Si significantly counteracted high Mn-elevated malondialdehyde and H₂O₂ concentrations and enhanced plant growth. In cv. ZLY, high Mn considerably raised SOD activities and glutathione concentrations, thus leading to relatively low oxidative damage.

Conclusions

Si-enhanced Mn tolerance was attributed mainly to restricted Mn transport in cv. XXY but to depressed Mn uptake in cv. ZLY. Silicon mainly influenced non-enzymatic antioxidants in these two rice cultivars under high Mn stress.     

Chlorophyll content and leaf elongation rate in wheat seedlings as a measure of manganese tolerance

After aluminum toxicity, manganese (Mn) toxicity is probably the second most important growth limiting factor in acid soils. The purpose of this study was to determine the feasibility of using chlorophyll content and leaf elongation rate (LER) for regrowth of Mn stressed seedlings as a rapid seedling based screening bioassay for Mn tolerance in segregating populations of wheat (Triticum aestivum L.). In one experiment, chlorophyll was determined for the cultivars Norquay (Mn-tolerant) and Columbus (Mn-sensitive) subjected to twelve Mn levels (2 to 2000 μM) in nutrient solutions. As Mn concentration increased, chlorophyll ‘a’ and ‘b’ contents of the Mn-tolerant cultivar decreased up to 9%, while in the Mn-sensitive cultivar it was reduced by as much as 43%. The chlorophyll ‘a/b’ ratio did not differ among Mn concentrations for either cultivar. In a second experiment, chlorophyll content and LER for regrowth of Mn stressed seedlings (1000 μM) was determined for Columbus and Katepwa (Mn-sensitive), Oslo (Mn-intermediate), and Norquay and Laura (Mn-tolerant). Manganese tolerance as assayed by chlorophyll ‘a’ and ‘b’ and LER was significantly correlated with Mn tolerance as assayed by the relative root weight methodology (RRW). Thus, chlorophyll content of Mn-stressed seedlings and LER of seedling regrowth appear to be suitable techniques for screening unreplicated selections of segregating populations for tolerance to Mn.     

Phenolics, lignin content and peroxidase activity in Picea omorika lines

We analyzed low molecular mass phenolics, lignin content and both soluble and cell wall bound peroxidase activity in the needles of three Picea omorika (Pancic) Purkyne lines grown in the generative seed orchard. The highest values of the total phenol content as well as of catechine, caffeic acid, coniferyl alcohol, isoferulic acid and lignin concentration were detected in B5 line (“semidichotomy” line). The soluble guaiacol peroxidase activity was the highest in A3 line (line “borealis”). The highest activity of cell wall bound peroxidases was measured in B5 line, and it was in correlation with lignin content.     

Composition and Properties of Hydrogen Peroxide Decomposing Systems in Extracellular and Total Extracts from Needles of Norway Spruce (Picea abies L., Karst.)

Hydrogen peroxide (H₂O₂) scavenging systems of spruce (Picea abies) needles were investigated in both extracts obtained from the extracellular space and extracts of total needles. As assessed by the lack of activity of symplastic marker enzymes, the extracellular washing fluid was free from intracellular contaminations. In the extracellular washing fluid ascorbate, glutathione, cysteine, and high specific activities of guaiacol peroxidases were observed. Guaiacol peroxidases in the extracellular washing fluid and needle homogenates had the same catalytic properties, i.e. temperature optimum at 50°C, pH optimum in the range of pH 5 to 6 and low affinity for guaiacol (apparent K_m = 40 millimolar) and H₂O₂ (apparent K_m = 1-3 millimolar). Needle homogenates contained ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase, glutathione reductase, and catalase, but not glutathione peroxidase activity. None of these activities was detected in the extracellular washing fluid. Ascorbate and glutathione related enzymes were freeze sensitive; ascorbate peroxidase was labile in the absence of ascorbate. The significance of extracellular antioxidants for the detoxification of injurious oxygen species is discussed.     

Lignin and Mn peroxidase-catalyzed oxidation of phenolic lignin oligomers

The oxidation of phenolic oligomers by lignin and manganese peroxidases was studied by transient-state kinetic methods. The reactivity of peroxidase intermediates compound I and compound II was studied with the phenol guaiacol along with a beta-O-4 phenolic dimer, trimer, and tetramer. Compound I of both peroxidases is much more reactive than compound II. The rate constants for these substrates with Mn peroxidase compound I range from 1.0 x 10(5) M-1 s-1 for guaiacol to 1.1 x 10(3) M-1 s-1 for the tetramer. Reactivity is much higher with lignin peroxidase compound I with rate constants ranging from 1.2 x 10(6) M-1s-1 for guaiacol to 3.6 x 10(5) M-1 s-1 for the tetramer. Rate constants with compound II are much lower with Mn peroxidase exhibiting very little reactivity. The rate constants dramatically decreased with both peroxidases as the size of the substrate increased. The extent of the decrease was much more dramatic with Mn peroxidase, leading us to conclude that, despite its ability to oxidize phenols, Mn2+ is the only physiologically significant substrate. The rate decrease associated with increasing substrate size was more gradual with lignin peroxidase. These data indicate that whereas Mn peroxidase cannot efficiently directly oxidize the lignin polymer, lignin peroxidase is well suited for direct oxidation of polymeric lignin.     

The Influence of Apoplastic Ascorbate on the Activities of Cell Wall-Associated Peroxidase and NADH Oxidase in Needles of Norway Spruce (Picea abies L.)

Cell wall-associated peroxidases (EC 1.11.1.7 [EC] ) were extractedfrom the current year's needles of Norway spruce trees (Piceaabies L.) in two fractions, namely soluble apoplastic peroxidasesand covalently wall-bound peroxidases. Peroxidase activitieswere determined with two substrates: coniferyl alcohol, whichis important for lignification, and NADH, which is necessaryfor the production of H₂O₂. Coniferyl alcohol peroxidase activitywas detected in both the soluble apoplastic fraction and thewall-bound fraction, whereas NADH oxidase activity was foundonly in the soluble apoplastic fraction. Net oxidation of coniferylalcohol and NADH was inhibited by ascorbate, which reduced theoxidized intermediates of the peroxidase- and oxidase-catalyzedreactions. Since ascorbate itself was oxidized in these reactions,the inhibition was not persistent and it was released once theascorbate present in the assay mixture had been oxidized. Ascorbatedelayed the oxidation of NADH 10-fold more efficiently thanthe oxidation of coniferyl alcohol. Although the level and theredox state of apoplastic ascorbate were lower in lignifyingneedles than in mature needles, the concentration, which was1.17 mM in apoplastic washing fluids, was sufficiently highto inhibit peroxidase activity in vitro. These results suggestthat peroxidases can catalyze lignification only if local differencesexist in the concentration of reduced ascorbate between lignifyingand non-lignifying tissues. (Received April 21, 1994; Accepted September 26, 1994)