Reactive oxygen species form part of a regulatory pathway initiating trans-differentiation of epidermal transfer cells in Vicia faba cotyledons

Various cell types can trans-differentiate to a transfer cell (TC) morphology characterized by deposition of polarized ingrowth walls comprised of a uniform layer on which wall ingrowths (WIs) develop. WIs form scaffolds supporting amplified plasma membrane areas enriched in transporters conferring a cellular capacity for high rates of nutrient exchange across apo- and symplasmic interfaces. The hypothesis that reactive oxygen species (ROS) are a component of the regulatory pathway inducing ingrowth wall formation was tested using Vicia faba cotyledons. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, on being placed into culture, their adaxial epidermal cells rapidly (hours) form ingrowth walls on their outer periclinal walls. These are readily visualized by electron microscopy, and epidermal peels of their trans-differentiating cells allow measures of cell-specific gene expression. Ingrowth wall formation responded inversely to pharmacological manipulation of ROS levels, indicating that a flavin-containing enzyme (NADPH oxidase) and superoxide dismutase cooperatively generate a regulatory H(2)O(2) signature. Extracellular H(2)O(2) fluxes peaked prior to the appearance of WIs and were followed by a slower rise in H(2)O(2) flux that occurred concomitantly, and co-localized, with ingrowth wall formation. De-localizing the H(2)O(2) signature caused a corresponding de-localization of cell wall deposition. Temporal and epidermal cell-specific expression profiles of VfrbohA and VfrbohC coincided with those of extracellular H(2)O(2) production and were regulated by cross-talk with ethylene. It is concluded that H(2)O(2) functions, downstream of ethylene, to activate cell wall biosynthesis and direct polarized deposition of a uniform wall on which WIs form.     

Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species

Several bacteria possess periplasmic Cu,Zn superoxide dismutases which can confer protection from extracellular reactive oxygen species. Thus, deletion of the sodC1 gene reduces Salmonella enterica serovar Typhimurium ability to colonize the spleens of wild type mice, but enhances virulence in p47phox mutant mice. To look into the role of periplamic Cu,Zn superoxide dismutase and into possible additive effects of the ferritin-like Dps protein involved in hydrogen peroxide detoxification, we have analyzed bacterial survival in response to extracellular sources of superoxide and/or hydrogen peroxide. Exposure to extracellular superoxide of Salmonella Typhimurium mutant strains lacking the sodC1 and sodC2 genes and/or the dps gene does not cause direct killing of bacteria, indicating that extracellular superoxide is poorly bactericidal. In contrast, all mutant strains display a sharp hydrogen peroxide-dependent loss of viability, the dps,sodC1,sodC2 mutant being less resistant than the dps or the sodC1,sodC2 mutants. These findings suggest that the role of Cu,Zn superoxide dismutase in bacteria is to remove rapidly superoxide from the periplasm to prevent its reaction with other reactive molecules. Moreover, the nearly additive effect of the sodC and dps mutations suggests that localization of antioxidant enzymes in different cellular compartments is required for bacterial resistance to extracytoplasmic oxidative attack.     

The distribution of cell wall polymers during antheridium development and spermatogenesis in the Charophycean green alga, Chara corallina

Background and Aims

The production of multicellular gametangia in green plants represents an early evolutionary development that is found today in all land plants and advanced clades of the Charophycean green algae. The processing of cell walls is an integral part of this morphogenesis yet very little is known about cell wall dynamics in early-divergent green plants such as the Charophycean green algae. This study represents a comprehensive analysis of antheridium development and spermatogenesis in the green alga, Chara corallina.

Methods

Microarrays of cell wall components and immunocytochemical methods were employed in order to analyse cell wall macromolecules during antheridium development.

Key Results

Cellulose and pectic homogalacturonan epitopes were detected throughout all cell types of the developing antheridium including the unique cell wall protuberances of the shield cells and the cell walls of sperm cell initials. Arabinogalactan protein epitopes were distributed only in the epidermal shield cell layers and anti-xyloglucan antibody binding was only observed in the capitulum region that initially yields the sperm filaments. During the terminal stage of sperm development, no cell wall polymers recognized by the probes employed were found on the scale-covered sperm cells.

Conclusions

Antheridium development in C. corallina is a rapid event that includes the production of cell walls that contain polymers similar to those found in land plants. While pectic and cellulosic epitopes are ubiquitous in the antheridium, the distribution of arabinogalactan protein and xyloglucan epitopes is restricted to specific zones. Spermatogenesis also includes a major switch in the production of extracellular matrix macromolecules from cell walls to scales, the latter being a primitive extracellular matrix characteristic of green plants.     

The EGCg-induced redox-sensitive activation of endothelial nitric oxide synthase and relaxation are critically dependent on hydroxyl moieties

Several rich sources of polyphenols stimulate the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase (eNOS). The present study examined the molecular mechanism underlying the stimulatory effect of epicatechins on eNOS. NO-mediated relaxation was assessed using porcine coronary artery rings in the presence of indomethacin, and charybdotoxin plus apamin, inhibitors of cyclooxygenases and EDHF-mediated responses, respectively. The phosphorylation level of Akt and eNOS was assessed in cultured coronary artery endothelial cells by Western blot, and ROS formation using dihydroethidine. (−)-Epigallocatechin-3-O-gallate (EGCg) caused endothelium-dependent relaxations in coronary artery rings and the phosphorylation of Akt and eNOS in endothelial cells. These responses were inhibited by membrane-permeant analogues of superoxide dismutase and catalase, whereas native superoxide dismutase, catalase and inhibitors of major enzymatic sources of reactive oxygen species including NADPH oxidase, xanthine oxidase, cytochrome P450 and the mitochondrial respiration chain were without effect. The EGCg derivative with all hydroxyl functions methylated induced neither relaxations nor the intracellular formation of ROS, whereas both responses were observed when the hydroxyl functions on the gallate moiety were present. In conclusion, EGCg causes endothelium-dependent NO-mediated relaxations of coronary artery rings through the Akt-dependent activation of eNOS in endothelial cells. This response is initiated by the intracellular formation of superoxide anions and hydrogen peroxide, and is critically dependent on the gallate moiety and on the presence of hydroxyl functions possibly through intracellular auto-oxidation.     

Protective effect of Ligusticum chuanxiong and Angelica sinensis on endothelial cell damage induced by hydrogen peroxide

Ligusticum chuanxiong and Angelica sinensis have been widely used in traditional Chinese medicine to treat some pathological settings such as atherosclerosis and hypertension. We determined the protective effect of the extract of Ligusticum chuanxiong and Angelica sinensis (ELCAS) on human umbilical vein endothelial cells (ECV304) damage induced by hydrogen peroxide. ECV304 cells were pre-treated with ELCAS and exposed to 5 mM hydrogen peroxide. The results show that ELCAS dose- and time-dependently protected ECV304 cells against hydrogen peroxide damage and suppressed the production of reactive oxygen species (ROS). The decrement of ROS may be associated with increased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Western blot analysis revealed that ELCAS significantly increased the phosphorylation of ERK and promoted eNOS expression. These observations indicate that ELCAS protected ECV304 cells against hydrogen peroxide damage by enhancing the antioxidative ability, activating ERK and eNOS signaling pathway. Our data also provide new evidence of Ligusticum chuanxiong and Angelica sinensis in preventing both cardiovascular and cerebrovascular diseases.     

Role of cellular superoxide dismutase against reactive oxygen metabolite injury in cultured bovine aortic endothelial cells.

We examined the protective effect of cellular superoxide dismutase against extracellular hydrogen peroxide in cultured bovine aortic endothelial cells. 51Cr-labeled cells were exposed to hydrogen peroxide generated by glucose oxidase/glucose. Glucose oxidase caused a dose-dependent increase of 51Cr release. Pretreatment with diethyldithiocarbamate enhanced injury induced by glucose oxidase, corresponding with the degree of inhibition of endogenous superoxide dismutase activity. Inhibition of cellular superoxide dismutase by diethyldithiocarbamate was not associated either with alteration of other antioxidant defenses or with potentiation of nonoxidant injury. Enhanced glucose oxidase damage by diethyldithiocarbamate was prevented by chelating cellular iron. Inhibition of cellular xanthine oxidase neither prevented lysis by hydrogen peroxide nor diminished enhanced susceptibility by diethyldithiocarbamate. These results suggest that, in cultured endothelial cells: 1) cellular superoxide is involved in mediating hydrogen peroxide-induced damage; 2) superoxide, which would be generated upon exposure to excess hydrogen peroxide independently of cellular xanthine oxidase, promotes the Haber-Weiss reaction by initiating reduction of stored iron (Fe3+) to Fe2+; 3) cellular iron catalyzes the production of a more toxic species from these two oxygen metabolites; 4) cellular superoxide dismutase plays a critical role in preventing hydrogen peroxide damage by scavenging superoxide and consequently by inhibiting the generation of the toxic species.     

Ultrastructure of fibre and parenchyma cell walls during early stages of culm development in Dendrocalamus asper

BACKGROUND AND AIMS: The anatomy of bamboo culms and the multilayered structure of fibre cell walls are known to be the main determinant factors for its physical and mechanical properties. Studies on the bamboo cell wall have focussed mainly on fully elongated and mature fibres. The main aim of this study was to describe the ultrastructure of primary and secondary cell walls in culm tissues of Dendrocalamus asper at different stages of development. METHODS: The development of fibre and parenchyma tissues was classified into four stages based on light microscopy observations made in tissues from juvenile plants. The stages were used as a basis for transmission electron microscopy study on the ultrastructure of the cell wall during the process of primary and early secondary cell wall formation. Macerations and phloroglucinol-HCl staining were employed to investigate fibre cell elongation and fibre cell wall lignification, respectively. KEY RESULTS: The observations indicated that the primary wall is formed by the deposition of two distinct layers during the elongation of the internode and that secondary wall synthesis may begin before the complete cessation of internode and fibre elongation. Elongation was followed by a maturation phase characterized by the deposition of multiple secondary wall layers, which varied in number according to the cell type, location in the culm tissue and stage of shoot development. Lignification of fibre cell walls started at the period prior to the cessation of internode elongation. CONCLUSIONS: The structure of the primary cell wall was comprised of two layers. The fibre secondary cell wall began to be laid down while the cells were still undergoing some elongation, suggesting that it may act to cause the slow-down and eventual cessation of cell elongation.     

Superoxide,hydrogen peroxide,and the respiratory burst of fungally infected plant cells

The principal route of oxygen utilization in the respiratory burst of fungally infected plants was determined from stoichiometries of the uptake and electronic reduction of oxygen in cotton cells exposed to Aspergillus favus walls. Using 2,2-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) and epinephrine as redox reagents to manipulate oxygen transitions, we found that oxygen consumption doubled when superoxide disproportionation was abolished and was abolished when disproportionation doubled. Of four possible pathways for oxygen consumption, only monovalent reduction of molecular oxygen to superoxide was consistent with this inversely proportional relationship. According to the observed rate of oxygen consumption in this pathway and in the absence of competition to disproportionation of superoxide, infected cells are capable of generating intracellular concentrations of 1 M hydrogen peroxide in 13 min.     

Silica nanoparticles aid in structural leaf coloration in the Malaysian tropical rainforest understorey herb Mapania caudata

Background and Aims

Blue-green iridescence in the tropical rainforest understorey sedge Mapania caudata creates structural coloration in its leaves through a novel photonic mechanism. Known structures in plants producing iridescent blues consist of altered cellulose layering within cell walls and in special bodies, and thylakoid membranes in specialized plastids. This study was undertaken in order to determine the origin of leaf iridescence in this plant with particular attention to nano-scale components contributing to this coloration.

Methods

Adaxial walls of leaf epidermal cells were characterized using high-pressure-frozen freeze-substituted specimens, which retain their native dimensions during observations using transmission and scanning microscopy, accompanied by energy-dispersive X-ray spectroscopy to identify the role of biogenic silica in wall-based iridescence. Biogenic silica was experimentally removed using aqueous Na₂CO₃ and optical properties were compared using spectral reflectance.

Key Results and Conclusions

Blue iridescence is produced in the adaxial epidermal cell wall, which contains helicoid lamellae. The blue iridescence from cell surfaces is left-circularly polarized. The position of the silica granules is entrained by the helicoid microfibrillar layers, and granules accumulate at a uniform position within the helicoids, contributing to the structure that produces the blue iridescence, as part of the unit cell responsible for 2 ° Bragg scatter. Removal of silica from the walls eliminated the blue colour. Addition of silica nanoparticles on existing cellulosic lamellae is a novel mechanism for adding structural colour in organisms.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 μmol quanta m^− 2 s^− 1 it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.     

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Background and Aims Plants regulate cellular oxygen partial pressures (pO₂), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO₂ regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.Methods The gradients in pO₂ from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO₂ production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.Key Results Steep internal pO₂ gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO₂ production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO₂ gradient showed a biphasic profile, where the minimum pO₂ was external to the core of the bud complex.Conclusions Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.     

ADP stimulates the respiratory burst without activation of ERK and AKT in rat alveolar macrophages

Alveolar macrophages (AM) are the first line of defense against infection in the lungs. We previously showed that the production of superoxide and hydrogen peroxide, i.e., the respiratory burst, is stimulated by adenine nucleotides (ADP > ATP) in rat AM through signaling pathways involving calcium and protein kinase C. Here, we further show that ADP induces a rapid increase in the tyrosine phosphorylation of several proteins that was reduced by the tyrosine kinase inhibitor genistein, which also inhibited the respiratory burst. Interestingly, ADP did not trigger the activation of the mitogen-activated protein kinases ERK1 and ERK2, or that of protein kinase B/AKT, a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway. This is in contrast to another stimulus of the respiratory burst, zymosan-activated serum (ZAS), which activates both the ERK and PI3K pathways. Thus, this study demonstrates that the receptor for ADP in rat AM is not coupled to the ERK and AKT pathways and, that neither the ERK pathway nor AKT is essential to induce the activation of the NAPDH oxidase by ADP in rat AM while tyrosine kinases appeared to be required. The rate and amount of hydrogen peroxide released by the ADP-stimulated respiratory burst was similar to that produced by ZAS stimulation. The absence of ERK activation after ADP stimulation therefore suggests that hydrogen peroxide is not sufficient to activate the ERK pathway in rat AM. Nonetheless, as hydrogen peroxide was necessary for ERK activation by ZAS, this indicates that, in contrast to ADP, ZAS stimulates a pathway that is targeted by hydrogen peroxide and leads to ERK activation.     

The topology of superoxide production by complex III and glycerol 3-phosphate dehydrogenase in Drosophila mitochondria

The topology of superoxide generation by sn-glycerol 3-phosphate dehydrogenase and complex III in intact Drosophila mitochondria was studied using aconitase inactivation to measure superoxide production in the matrix, and hydrogen peroxide formation in the presence of superoxide dismutase to measure superoxide production from both sides of the membrane. Aconitase inactivation was calibrated using the known rate of matrix superoxide production from complex I. Glycerol phosphate dehydrogenase generated superoxide about equally to each side of the membrane, whereas centre o of complex III in the presence of antimycin A generated superoxide about 30% on the cytosolic side and 70% on the matrix side.     

New insights into pioneer root xylem development: evidence obtained from Populus trichocarpa plants grown under field conditions

Background and Aims

Effective programmed xylogenesis is critical to the structural framework of the plant root system and its central role in the acquisition and long-distance transport of water and nutrients. The process of xylem differentiation in pioneer roots under field conditions is poorly understood. In this study it is hypothesized that xylogenesis, an example of developmental programmed cell death (PCD), in the roots of woody plants demonstrates a clearly defined sequence of events resulting in cell death. A comprehensive analysis was therefore undertaken to identify the stages of xylogenesis in pioneer roots from procambial cells to fully functional vessels with lignified cell walls and secondary cell wall thickenings.

Methods

Xylem differentiation was monitored in the pioneer roots of Populus trichocarpa at the cytological level using rhizotrons under field conditions. Detection and localization of the signalling molecule nitric oxide (NO) and hydrogen peroxide (H₂O₂) was undertaken and a detailed examination of nuclear changes during xylogenesis was conducted. In addition, analyses of the expression of genes involved in secondary cell wall synthesis were performed in situ.

Key Results

The primary event in initially differentiating tracheary elements (TEs) was a burst of NO in thin-walled cells, followed by H₂O₂ synthesis and the appearance of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei. The first changes in nuclear structure were observed in the early stages of xylogenesis of pioneer roots, prior to lignification; however, the nucleus was detectable under transmission electron microscopy in differentiating cells until the stage at which vacuole integrity was maintained, indicating that their degradation was slow and prolonged. The subsequent sequence of events involved secondary cell wall formation and autophagy. Potential gene markers from the cinnamyl alcohol dehydrogenase (CAD) gene family that were related to secondary wall synthesis were associated with primary xylogenesis, showing clear expression in cells that undergo differentiation into TEs and in the thin-walled cells adjacent to the xylem pole.

Conclusions

The early events of TE formation during pioneer root development are described, together with the timing of xylogenesis from signalling via NO, through secondary cell wall synthesis and autophagy events that are initiated long before lignification. This is the first work describing experiments conducted in planta on roots under field conditions demonstrating that the process of xylogenesis in vivo might be gradual and complex.     

In vitro superoxide activity in the haemolymph of the West Indian leaf cockroach,Blaberus discoidalis

The respiratory burst is an NADPH oxidase-driven reduction of molecular oxygen to superoxide, which can occur in phagocytic cells as part of an antimicrobial defence, and is well documented among the vertebrates. This paper describes a process resembling the respiratory burst, which occurs in the haemolymph and haemocytes of the cockroach, Blaberus discoidalis. The in vitro reduction of nitroblue tetrazolium by superoxide to formazan was measured spectrophotometrically in B. discoidalis haemolymph in response to various immune elicitors. Nitroblue tetrazolium reduction was partly impeded in the presence of superoxide dismutase, a specific antioxidant which converts superoxide to hydrogen peroxide, as well as by chemicals known to inhibit the respiratory burst in vertebrates (trifluoperazine, diphenylene iodonium, and N-ethylmaleimide). This suggests the generation of superoxide anions by haemolymph as part of an immune response. Furthermore, formazan staining of elicitor-treated haemocytes was observed microscopically, with less intense staining in the presence of superoxide dismutase. Finally, respiratory burst inhibitors and superoxide dismutase enhanced the growth of E. coli incubated in whole haemolymph, implying a role for haemolymph-derived superoxide in antibacterial defence.     

A bentazone-resistant mutant of cyanobacterium,Synechococcus elongatus PCC7942 adapts different strategies to counteract on bromoxynil- and salt-mediated oxidative stress

A bentazone-resistant mutant of Synechococcus elongatus PCC7942, called Mu2, tolerated elevated NaCl concentrations. As bentazone and bromoxynil exhibit similar mechanism of action, we investigated whether the mutant also toleratedbromoxynil and found it to be true. The line of investigation was then whether the acclimation strategy for the three stressors, bentazone, bromoxynil and NaCl was same or different. The cellular contents of malondialdehyde, hydrogen peroxide and superoxide increased in wild type strain following all the treatments suggesting their toxicities due to oxidative response. Notwithstanding, there were apparently different anti-oxidative measures pertaining to the herbicide and salinity stress. Glutathione contents and activities of superoxide dismutase, catalase-peroxidase, glutathione S-transferase and glutathione reductase decreased under NaCl, whereas bromoxynil affected only glutathione S-transferase reductase. Moreover, in-gel assays revealed that bromoxynil promoted appearance of isozymes of catalase-peroxidase, while NaCl induced such response only for superoxide dismutase. On the other hand, in Mu2, glutathione peroxidase-reductase and glutathione showed upward trend after bromoxynil exposure, whereas NaCl raised peroxidase and superoxide dismutase. Proteome comparison revealed peroxiredoxin Q to be highly expressed in wild type strain under bromoxynil, whereas NaCl favoured flavodoxin over-expression. Their amounts were already high in Mu2. We suggest that Mu2 acclimatized to bromoxynil in a manner similar to bentazone by upgrading peroxiredoxin Q and glutathione peroxidase-reductase. Conversely, for NaCl it devised another mechanism involving peroxidase and superoxide dismutase, and flavodoxin.     

Possible functions of extracellular peroxidases in stress-induced generation and detoxification of active oxygen species

Extracellular peroxidases are classified as free, or ionically or covalently bound to the cell wall. In addition, peroxidase-like activities have often been demonstrated at the outer surface of protoplasts and plasma membrane preparations. Under certain conditions apoplastic peroxidases have been shown to contribute to the formation of superoxide and hydrogen peroxide during the `oxidative burst' through the oxidation of a reductant. However, the identity of this reductant remains unclear. It has been suggested that the production of these active oxygen species may play important roles in plant responses to biotic and abiotic stress. Extracellular release of pre-existing and de novo synthesis of apoplastic peroxidases is regulated by changing environmental conditions. While the oxidative burst could potentially be harmful to a plant's own cells, tissues can rapidly metabolize even high concentrations of hydrogen peroxide. Recent work has shown that when extracellular hydrogen peroxide exceeds the supplies of reductants, class II and class III peroxidases can display catalase-like activity. Under these conditions, hydrogen peroxide is able to act as both oxidizing and reducing substrate. It seems likely therefore, that a further role of extracellular peroxidases is to protect plants from the consequences of the oxidative burst that they themselves are responsible for producing.     

Cell-wall-bound oxidases from tobacco (Nicotiana tabacum) xylem participate in lignin formation

A band of cells closest to the cambium in the xylem of tobacco (Nicotiana tabacum L. cv. Samsun) stems oxidized 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS), o-dianisidine and syringaldazine in the absence of exogenously added hydrogen peroxide. The oxidation was not prevented by catalase which suggests that the oxidation is not dependent on the production and utilisation of endogenous hydrogen peroxide by cell-wall peroxidases. Cell walls, isolated from tobacco xylem, also oxidized these substrates in the absence of added hydrogen peroxide. The cell walls consumed molecular oxygen whilst oxidizing a range of compounds including coniferyl alcohol. The substrate preference and sensitivity to inhibitors suggest the presence of laccasetype polyphenol oxidases (p-diphenol:O₂ oxidoreductase EC 1.14.18.1) which are covalently bound to the wall. The oxidation of coniferyl alcohol by the xylem cell walls was confirmed by assays based on the disappearance of coniferyl alcohol and was not affected by the presence of 500 units·mi^-1 catalase or Superoxide dismutase. Prolonged incubation of cell walls with coniferyl alcohol led to the production of a yellow-orange water-insoluble material that precipitated with the cell walls. Although a proportion of this material was soluble in methanol, the majority was tightly associated with the cell walls. These coloured cell walls had elevated lignin contents when assayed by the acetyl-bromide method. Fourier transforminfrared spectroscopic analysis of the coloured cell walls indicated that the increased lignin content is due to the deposition of guaiacyl-type lignin. Digestion of the xylem cell walls with Driselase, a mixture of fungal glycases, produced a wall residue that had a dramatically reduced ability to oxidize ABTS in the absence of added H₂O₂. However, oxidase activity could not be detected in the Driselase-solubilized extract, although small amounts of oxidase activity could be recovered from the Driselaseresistant wall residue by extraction in 3 M CaCl₂.Abbreviations ABTS 2,2-azinobis-(3-ethylbenzo-thiazoline-6-sulphonate) - dl-DOPA 3-(3,4-dihydroxyphenyl)-alanine - FTIR Fourier transform infra-red - o-D o-dianisidine - o-pD o-phenylenediamine - SYR syringaldazine The authors acknowledge funding from the Scottish Office Agriculture and Food Department. They would like to thank Professor J.R. Hillman for his support, Dr. G.D. Lyon for his help and advice with the oxygen electrode and Mrs F. Carr for lignin determinations.     

Oxidase, superoxide dismutase, and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs

Methanobactin (mb) is a copper-binding chromopeptide that appears to be involved in oxidation of methane by the membrane-associated or particulate methane monooxygenase (pMMO). To examine this potential physiological role, the redox and catalytic properties of mb from three different methanotrophs were examined in the absence and presence of O₂. Metal free mb from the type II methanotroph Methylosinus trichosporium OB3b, but not from the type I methanotrophs Methylococcus capsulatus Bath or Methylomicrobium album BG8, were reduced by a variety of reductants, including NADH and duroquinol, and catalyzed the reduction of O₂ to . Copper-containing mb (Cu-mb) from all three methanotrophs showed several interesting properties, including reductase dependent oxidase activity, dismutation of to H₂O₂, and the reductant dependent reduction of H₂O₂ to H₂O. The superoxide dismutase-like and hydrogen peroxide reductase activities of Cu-mb were 4 and 1 order(s) of magnitude higher, respectively, than the observed oxidase activity. The results demonstrate that Cu-mb from all three methanotrophs are redox-active molecules and oxygen radical scavengers, with the capacity to detoxify both superoxide and hydrogen peroxide without the formation of the hydroxyl radicals associated with Fenton reactions. As previously observed with Cu-mb from Ms. trichosporium OB3b, Cu-mb from both type I methanotrophs stimulated pMMO activity. However, in contrast to previous studies using mb from Ms. trichosporium OB3b, pMMO activity was not inhibited by mb from the two type I methanotrophs at low copper to mb ratios.