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1.
BACKGROUND AND AIMS: Following previous findings of high extracellular redox activity in lichens and the presence of laccases in lichen cell walls, the work presented here additionally demonstrates the presence of tyrosinases. Tests were made for the presence of tyrosinases in 40 species of lichens, and from selected species their cellular location and molecular weights were determined. The effects of stress and inhibitors on enzyme activity were also studied. METHODS: Tyrosinase and laccase activities were assayed spectrophotometrically using a variety of substrates. The molecular mass of the enzymes was estimated using polyacrylamide gel electrophoresis. KEY RESULTS: Extracellular tyrosinase and laccase activity was measured in 40 species of lichens from different taxonomic groupings and contrasting habitats. Out of 20 species tested from the sub-order Peltigerineae, all displayed significant tyrosinase and laccase activity, while activity was low or absent in other species tested. Representatives from both groups of lichens displayed low peroxidase activities. Identification of the enzymes as tyrosinases was confirmed by the ability of lichen thalli or leachates derived by shaking lichens in distilled water to metabolize substrates such as L-dihydroxyphenylalanine (DOPA), tyrosine and epinephrine readily in the absence of hydrogen peroxide, the sensitivity of the enzymes to the inhibitors cyanide, azide and hexylresorcinol, activation by SDS and having typical tyrosinase molecular masses of approx. 60 kDa. Comparing different species within the Peltigerineae showed that the activities of tyrosinases and laccase were correlated to each other. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. CONCLUSIONS: Cell walls of lichens in sub-order Peltigerineae have much higher activities and a greater diversity of cell wall redox enzymes compared with other lichens. Possible roles of tyrosinases include melanization, removal of toxic phenols or quinones, and production of herbivore deterrents.  相似文献   

2.
Rates of extracellular superoxide radical (O2· ?) formation were measured in 34 species of lichens from different taxonomic groupings and contrasting habitats before and after desiccation stress. All 21 species from the suborder Peltigerineae produce O2· ? extracellularly at high rates, even when they are not stressed. In addition, some species show a burst of O2· ? production during rehydration following desiccation. Extracellular production of O2· ? is almost absent in the species from other lichen groups. In general, production of high levels of O2· ? and the existence of an inducible oxidative burst are best developed in species growing in wet microhabitats. Rates of O2· ? production are also positively correlated to previously published indices of lichen metabolic activity. Preliminary studies on the identity of the O2· ? producing enzymes suggest that they do not possess the classical characteristics of those suggested to produce reactive oxygen species in higher plants. Patterns of O2· ? production are discussed in terms of the strategies used by different lichens groups in their defence against pathogenic fungi and bacteria.  相似文献   

3.
4.
It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O2•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O2•− in a hydrogen peroxide (H2O2)-dependent manner. However, this model is incomplete since the source of the initially required H2O2 could not be explained. Based on the recently proposed role for H2O2-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA.  相似文献   

5.
Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H2O2 modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H2O2 in different light qualities. H2O2 either promoted germination or repressed germination depending on the light wavelengths, showing that H2O2 acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H2O2-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.  相似文献   

6.
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 Km = 40 millimolar) and H2O2 (apparent Km = 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.  相似文献   

7.
Vitiligo is an autoimmune disease characterized by depigmentation. Kaempferol is a flavonoid compound with broad anti-inflammatory and antioxidant properties. The purpose of this study was to investigate the effect of kaempferol on melanogenesis in PIG1 normal human skin melanocytes and its response to oxidative stress. The effect of kaempferol on melanin synthesis in PIG1 normal human skin melanocytes was explored by measuring tyrosinase activity, melanin content, mRNA and protein expression of key enzymes and expression of related pathway proteins. The effects of kaempferol pretreatment on cell viability, apoptosis, ROS level and HO-1 protein level under H2O2 stimulation were explored. When treated with kaempferol, the tyrosinase activity and melanin content of PIG1 cells increased, the mRNA and protein expressions of TYR, TRP1, TRP2 and MITF increased, and the phosphorylation level of ERK1/2 increased. Upon the stimulation of H2O2, kaempferol reduced the production of ROS, decreased apoptosis and increased the protein expression of HO-1 in PIG1 cells. In addition, kaempferol inhibited oxidative stress-induced melanin reduction and promoted melanin synthesis in PIG1 cells and protected against H2O2-induced oxidative stress damage.  相似文献   

8.
A psychrotolerant and H2O2-resistant bacterium, Exiguobacterium oxidotolerans T-2-2T, exhibits extraordinary H2O2 resistance and produces catalase not only intracellularly but also extracellularly. The intracellular and extracellular catalases exhibited the same enzymatic characteristics, that is, they exhibited the temperature-dependent activity characteristic of a cold-adapted enzyme, their heat stabilities were similar to those of mesophilic enzymes and very high catalytic intensity. In addition, catalase gene analysis indicated that the bacterium possessed the sole clade 1 catalase gene corresponding to intracellular catalase. Hence, intracellular catalase is secreted into the extracellular space. In addition to intracellular and extracellular catalases, the inner circumference of the cells showed the localization of catalase in the mid-stationary growth phase, which was observed by immunoelectron microscopy using an antibody against the intracellular catalase of the strain. The cells demonstrated higher catalase activity in the mid-stationary growth phase than in the exponential growth phase. The catalase localized in the inner circumference can be dissociated by treatment with Tween 60. Thus, the localized catalase is not tightly bound to the inner circumference of the cells and may play a role in the oxidative defense of the cells under low metabolic state.  相似文献   

9.
We have investigated the physiological functions of the rapid generation of reactive oxygen species (ROS) and the implication of the antioxidant enzymes in the apoplast and symplast of roots of sunflower (Helianthus annuus L.) seedlings exposed to methyl jasmonate (MeJA, 50 μM). MeJA-elicited roots showed a fast increase in ROS content, followed by a marked increase in the activity of H2O2-scavenging enzymes, guaiacol peroxidase (GPX), ascorbate peroxidase (APX) and catalase (CAT). The mechanisms responsible for MeJA-induced H2O2 accumulation was investigated further by studying both the production and scavenging of H2O2 in the extracellular matrix. Peroxidases active against (2,2′-azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid], ABTS) and guaiacol were found in the apoplastic fluid, and proved to be ionically and covalently associated with sunflower cell walls, although only the peroxidase activities of the soluble apoplastic fractions and those ionically linked to the cell wall were correlated with the accumulation of the H2O2 detected. The results indicated that H2O2 accumulation is a complex and highly regulated event requiring the time-dependent stimulation and down-regulation of differently located enzymes, some of which are involved in H2O2 generation and degradation. It is concluded that exogenous MeJA may be involved in the oxidative stress processes by regulating antioxidant enzyme activities.  相似文献   

10.
Chen Q  Yang L  Ahmad P  Wan X  Hu X 《Planta》2011,233(3):583-592
Tea seed is believed to be recalcitrant based on its sensitivity to chilling or drying stress. Reactive oxygen species (ROS) and alterations in cytosolic redox status have been implicated in intolerance to desiccation by recalcitrant seed, but there is little information available regarding how ROS are regulated in seeds susceptible to drying stress. We investigated changes in protein expression and activity in tea embryo in response to desiccation using physiological and proteomic methods. Results showed that desiccation treatment dramatically induced the accumulation of H2O2 in tea embryos, accompanied by increased activities of antioxidant enzymes like ascorbate peroxidase (APX) and superoxide dismutase (SOD). Proteomic analyses also demonstrated that 23 proteins associated with defense response, metabolism and redox status were up-regulated following desiccation. Increase in antioxidants, ascorbic acid (AsA) and catalase (CAT) (H2O2 scavengers) partially assuaged desiccation damage to tea seed, resulting in improved germination rates. Higher accumulation of H2O2 aggravated desiccation damage to seeds leading to lower germination activity. We propose that desiccation causes an over-accumulation of ROS that are not efficiently scavenged by increased levels of antioxidant enzymes. High levels of ROS alter the redox status and are detrimental to seed viability. Reducing ROS to appropriate concentrations is an efficient way to reduce desiccation damage and improve germination rates of recalcitrant seeds.  相似文献   

11.
Changes in activities of the enzymes performing direct antioxidant functions were studied in 7–8-week-old plants Arabidopsis thaliana Heinh (L.) of Columbia (Col-0) ecotype. It was found that 5-day cold hardening at 2°C increased plant cold resistance to the subsequent stronger cooling. Under these conditions, the marked changes occurred in activities of superoxide dismutase and III type (guaiacol) peroxidses but not in that of catalase. The total peroxidase activity exceeded the catalase activity before cold hardening. Therefore, peroxidases are able to decompose more H2O2 than catalases and appear to make the dominant contribution to the protection from the cold damage.  相似文献   

12.
Endothelial cells are critical targets in both hypoxia-and reoxygenation-mediated lung injury. Reactive O2 species (ROS) have been implicated in the pathogenesis of hypoxic and reoxygenation lung injury, and xanthine dehydrogenase/oxidase (XDH/XO) is a major generator of the ROS. Porcine pulmonary artery endothelial cells (PAEC) have no detectable XDH/XO. This study was undertaken to examine (1) ROS production by hypoxic porcine PAEC and their mitochondria and (2) ROS production and injury in reoxygenated PAEC lacking XDH/XO activity. Intracellular H2O2 generation and extracellular H2O2 and O/2 release were measured after exposure to normoxia (room air-5% CO2), hypoxia (0% O2 -95% N-5% CO2), or hypoxia followed by normoxia or hyperoxia (95% O2-5% CO2). Exposure to hypoxia results in significant reductions in intracellular H2 O2 formation and extracellular release of H2 O2 and O2 by PAEC and mitochondria. The reductions occur with as little as a 2 h exposure and progress with continued exposure. During reoxygenation, cytotoxicity was not observed, and the production of ROS by PAEC and their mitochondria never exceeded levels observed in normoxic cells. The absence of XDH/XO may prevent porcine PAEC from developing injury and increased ROS production during reoxygenation. © 1995 Wiley-Liss, Inc.  相似文献   

13.
Lichens produce various oxidoreductases including heme-containing peroxidases and the copper-containing phenol oxidases tyrosinase and laccase. Our earlier findings suggested that significant oxidoreductase activity occurs mainly in lichens from the order Peltigerales. Here we show that the non-Peltigeralean lichen Usnea can display significant activities of peroxidases and laccases. Strong evidence for the involvement of peroxidases and laccases in saprotrophic activities comes from the observation that their activities are induced by “starvation” due to prolonged dark storage, and also by treatment with soluble cellulose and lignin breakdown products. We also show that, given a quinone and chelated Fe, Usnea can produce hydroxyl radicals; these radicals contribute to the break down of carbohydrates or lignin. However, hydroxyl radical production is independent of laccase and peroxidase activity. Laccases and peroxidases are involved in other aspects of lichen biology; here we show that peroxidases, but not laccases, can break down lichen substances. Reduction in the amounts of lichen substances will reduce photoprotection, which will increase the photosynthetic capacity of thalli during winter when light intensities are low.  相似文献   

14.
《Biotechnology advances》2017,35(6):815-831
Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly “fueling” electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.  相似文献   

15.
In plants, the chloroplast is the main reactive oxygen species (ROS) producing site under high light stress. Catalase (CAT), which decomposes hydrogen peroxide (H2O2), is one of the controlling enzymes that maintains leaf redox homeostasis. The catalase mutants with reduced leaf catalase activity from different plant species exhibit an H2O2‐induced leaf cell death phenotype. This phenotype was differently affected by light intensity or photoperiod, which may be caused by plant species, leaf redox status or growth conditions. In the rice CAT mutant nitric oxide excess 1 (noe1), higher H2O2 levels induced the generation of nitric oxide (NO) and higher S‐nitrosothiol (SNO) levels, suggesting that NO acts as an important endogenous mediator in H2O2‐induced leaf cell death. As a free radical, NO could also react with other intracellular and extracellular targets and form a series of related molecules, collectively called reactive nitrogen species (RNS). Recent studies have revealed that both RNS and ROS are important partners in plant leaf cell death. Here, we summarize the recent progress on H2O2‐induced leaf cell death and the crosstalk of RNS and ROS signals in the plant hypersensitive response (HR), leaf senescence, and other forms of leaf cell death triggered by diverse environmental conditions. [ Chengcai Chu (Corresponding author)]  相似文献   

16.
The life cycle of the plant pathogen Erwinia amylovora comprises periods inside and outside the host in which it faces oxidative stress caused by hydrogen peroxide (H2O2) and other compounds. The sources of this stress are plant defences, other microorganisms and/or exposure to starvation or other environmental challenges. However, the functional roles of H2O2‐neutralizing enzymes, such as catalases, during plant–pathogen interactions and/or under starvation conditions in phytopathogens of the family Erwiniaceae or closely related families have not yet been investigated. In this work, the contribution of E. amylovora catalases KatA and KatG to virulence and survival in non‐host environments was determined using catalase gene mutants and expression, as well as catalase activity analyses. The participation of E. amylovora exopolysaccharides (EPSs) in oxidative stress protection was also investigated. Our study revealed the following: (i) a different growth phase regulation of each catalase, with an induction by H2O2 and host tissues; (ii) the significant role of E. amylovora catalases as virulence and survival factors during plant–pathogen interactions; (iii) the induction of EPSs by H2O2 despite the fact that apparently they do not contribute to protection against this compound; and (iv) the participation of both catalases in the detoxification of the starvation‐induced intracellular oxidative stress, favouring the maintenance of culturability, and hence delaying the development of the viable but non‐culturable (VBNC) response.  相似文献   

17.
Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low‐potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H2O2 that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H2O2 in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H2O2, while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non‐scavenging mutant we verified that endogenous H2O2 formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H2O2 was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.  相似文献   

18.
Mitochondrial reactive oxygen species (ROS) play an important role in both physiological cell signaling processes and numerous pathological states, including neurodegenerative disorders such as Parkinson disease. While mitochondria are considered the major cellular source of ROS, their role in ROS removal remains largely unknown. Using polarographic methods for real-time detection of steady-state H2O2 levels, we were able to quantitatively measure the contributions of potential systems toward H2O2 removal by brain mitochondria. Isolated rat brain mitochondria showed significant rates of exogenous H2O2 removal (9–12 nmol/min/mg of protein) in the presence of substrates, indicating a respiration-dependent process. Glutathione systems showed only minimal contributions: 25% decrease with glutathione reductase inhibition and no effect by glutathione peroxidase inhibition. In contrast, inhibitors of thioredoxin reductase, including auranofin and 1-chloro-2,4-dinitrobenzene, attenuated H2O2 removal rates in mitochondria by 80%. Furthermore, a 50% decrease in H2O2 removal was observed following oxidation of peroxiredoxin. Differential oxidation of glutathione or thioredoxin proteins by copper (II) or arsenite, respectively, provided further support for the thioredoxin/peroxiredoxin system as the major contributor to mitochondrial H2O2 removal. Inhibition of the thioredoxin system exacerbated mitochondrial H2O2 production by the redox cycling agent, paraquat. Additionally, decreases in H2O2 removal were observed in intact dopaminergic neurons with thioredoxin reductase inhibition, implicating this mechanism in whole cell systems. Therefore, in addition to their recognized role in ROS production, mitochondria also remove ROS. These findings implicate respiration- and thioredoxin-dependent ROS removal as a potentially important mitochondrial function that may contribute to physiological and pathological processes in the brain.  相似文献   

19.
The complete apoplastic enzymatic antioxidant system, composed by class I ascorbate peroxidases (class I APXs), class III ascorbate peroxidases (class III APXs), ascorbate oxidases (AAOs), and other class III peroxidases (PRX), of wood-forming tissues has been studied in Populus alba, Citrus aurantium, and Eucalyptus camaldulensis. The aim was to ascertain whether these enzymatic systems may regulate directly (in the case of APXs), or indirectly (in the case of AAOs), apoplastic H2O2 levels in lignifying tissues, whose capacity to produce and to accumulate H2O2 is demonstrated here. Although class I APXs are particularly found in the apoplastic fraction of P. alba (poplar), and class III APXs are particularly found in the apoplastic fraction of C. aurantium (bitter orange tree), the results showed that the universal presence of AAO in the extracellular cell wall matrix of these woody species provokes the partial or total dysfunction of apoplastic class I and class III APXs, and of the whole plethora of non-enzymatic redox shuttles in which ascorbic acid (ASC) is involved, by the competitive and effective removal of ASC. In fact, the redox state (ASC/ASC+DHA) in intercellular wash fluids (IWFs) of these woody species was zero, and thus strongly shifted towards DHA (dehydroascorbate), the oxidized product of ASC. This imbalance of the apoplastic antioxidant enzymatic system apparently results in the accumulation of H2O2 in the apoplast of secondary wood-forming tissues, as can be experimentally observed. Furthermore, it is hypothesized that since AAO uses O2 to remove ASC, it could regulate O2 availability in the lignifying xylem and, thorough this mechanism, AAO could also control the activity of NADPH oxidase (the enzyme responsible for H2O2 production in lignifying tissues) at substrate level, by controlling the tension of O2. That is, the presence of AAO in the extracellular cell wall matrix appears to be essential for finely tuning the oxidative performance of secondary wood-forming tissues.  相似文献   

20.
Peroxidases have more functions than a Swiss army knife   总被引:38,自引:0,他引:38  
Plant peroxidases (class III peroxidases) are present in all land plants. They are members of a large multigenic family. Probably due to this high number of isoforms, and to a very heterogeneous regulation of their expression, plant peroxidases are involved in a broad range of physiological processes all along the plant life cycle. Due to two possible catalytic cycles, peroxidative and hydroxylic, peroxidases can generate reactive oxygen species (ROS) (OH, HOO), polymerise cell wall compounds, and regulate H2O2 levels. By modulating their activity and expression following internal and external stimuli, peroxidases are prevalent at every stage of plant growth, including the demands that the plant meets in stressful conditions. These multifunctional enzymes can build a rigid wall or produce ROS to make it more flexible; they can prevent biological and chemical attacks by raising physical barriers or by counterattacking with a large production of ROS; they can be involved in a more peaceful symbiosis. They are finally present from the first hours of a plants life until its last moments. Although some functions look paradoxical, the whole process is probably regulated by a fine-tuning that has yet to be elucidated. This review will discuss the factors that can influence this delicate balance.  相似文献   

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