Enzymatic Reactive Oxygen Species Scavenging Research Articles

The biocontrol potentiality of Bacillus amyloliquefaciens against postharvest soft rot of tomatoes and insights into the underlying mechanisms

Tomatoes can easily be affected by soft rot, one of the main postharvest infections caused by a bacterium called Pectobacterium carotovorum. This study examined the soft rot control effectiveness by Bacillus amyloliquefaciens in tomatoes, and identify the underlying action mechanisms from the perspective of the inhibition effects of antibacterial metabolites derived from B. amyloliquefaciens on pathogen and the influences of this antagonist on reactive oxygen species (ROS) scavenging capability of the fruit. The results demonstrated that B. amyloliquefaciens effectively lessened postharvest soft rot occurrence in tomatoes. The non-volatile metabolites synthesized by B. amyloliquefaciens suppressed P. carotovorum subsp. brasiliense (Pcb) growth in vitro and in vivo. The antibacterial ingredients presented in methanol-soluble sediments obtained from non-volatile metabolites. UHPLC-ESI-MS spectra revealed that the primary antibacterial ingredients were C12-C15 surfactin analogues, along with C14-C16 iturins and C16-C17 fengycins. Moreover, B. amyloliquefaciens could raise the activity of enzymatic ROS scavenging system in tomatoes, including several antioxidases and the enzymes related to glutathione peroxidase (GPX) and ascorbate-glutathione (AsA-GSH) cycles. In addition, B. amyloliquefaciens increased the levels of non-enzymatic system for ROS scavenging, including AsA and GSH. Together, O2-·production rate, H2O2 and malondialdehyde (MDA) levels were reduced. It suggested that B. amyloliquefaciens enhanced the ROS scavenging ability of tomatoes, thereby improving their resistance to the pathogen.

High-nitrogen fertilizer alleviated adverse effects of drought stress on the growth and photosynthetic characteristics of Hosta ‘Guacamole’

BackgroundSeveral plants are facing drought stress due to climate change in recent years. In this study, we aimed to explore the effect of varying watering frequency on the growth and photosynthetic characteristics of Hosta ‘Guacamole’. Moreover, we investigated the effect of high-nitrogen and -potassium fertilizers on alleviating the impacts of drought stress on the morphology, photosynthetic characteristics, chlorophyll fluorescence, fast chlorophyll a fluorescence transient, JIP-test parameters, and enzymatic and non-enzymatic scavenging system for reactive oxygen species (ROS) in this species.ResultsLeaf senescence, decreased chlorophyll contents, limited leaf area, and reduced photosynthetic characteristics and oxygen-evolving complex (OEC) activity were observed in Hosta ‘Guacamole’ under drought stress. However, high-nitrogen fertilizer (30-10-10) could efficiently alleviate and prevent the adverse effects of drought stress. High-nitrogen fertilizer significantly increased chlorophyll contents, which was higher by 106% than drought stress. Additionally, high-nitrogen fertilizer significantly improved net photosynthetic rate and water use efficiency, which were higher by 467% and 2900% than those under drought stress. It attributes that high-nitrogen fertilizer could reduce transpiration rate of leaf cells and stomatal opening size in drought stress. On the other hand, high-nitrogen fertilizer enhanced actual photochemical efficiency of PS II and photochemical quenching coefficient, and actual photochemical efficiency of PS II significantly higher by 177% than that under drought stress. Furthermore, high-nitrogen fertilizer significantly activated OEC and ascorbate peroxidase activities, and enhanced the performance of photosystem II and photosynthetic capacity compared with high-potassium fertilizers (15-10-30).ConclusionsHigh-nitrogen fertilizer (30-10-10) could efficiently alleviate the adverse effects of drought stress in Hosta ‘Guacamole’ via enhancing OEC activity and photosynthetic performance and stimulating enzymatic ROS scavenging system.

Colonization potential of endophytes from halophytic plants growing in the "Runn of Kutch" salt marshes and their contribution to mitigating salt stress in tomato cultivation.

Increasing soil salinity depreciates the quantity of the crop produce. Looking at the tremendous potential of plant-associated microorganisms in salinity stress mitigation, it would be very useful in exploring and deciphering salt-tolerant microorganisms from halophytic plants and their utilization in cultivated plants. With this aim, in the present study, four halophytic plants were taken from Rann of Kutch, and bacterial endophytes were isolated from different plant organs. These endophytes were characterized by plant growth and health promotion features. The molecular identification was done based on 16 s rRNA sequence similarity. It was found that the endophytic bacteria isolated from 4 different halophytes found sharing phylogenetic relatedness. Four potential endophytes Alkalihalobacillus gibsonii 2H2, Achromobacter insuavis 2H18, Terribacillus halophilus 2H20, and Bacillus siamensis 4H1 were tested in tomato for salinity stress alleviation. Changes in the levels of antioxidants were analyzed. Total chlorophyll, total phenolics, malondialdehyde, and proline content indicated reduced damage in the plant system due to salinity by the application of endophytes. All the treatments exhibited low levels of electrolyte leakage. The accumulation of enzymatic reactive oxygen species scavengers was assessed from the levels of peroxidase, catalase, superoxide dismutase, phenylalanine ammonia-lyase, ascorbate peroxidase, and guiacol peroxidase. The NBT and DAB staining confirmed the findings. The reduction in the accumulation of Na+ ions in tomato leaves was visualized using Sodium Green probes under CSLM and found to be lowest in Terribacillus halophilus 2H20 and Bacillus siamensis 4H1 inoculated plants. The endophyte Terribacillus halophilus 2H20 was the most promising isolate. The colonization in tomato roots was confirmed using a cell tracker system. Results showed that the endophytes were found to have salinity stress mitigation traits. The efficiency could be further improved with the combination of other endophytes tested earlier.

Ascorbate peroxidase plays an important role in photoacclimation in the extremophilic red alga Cyanidiococcus yangmingshanensis.

Acidothermophilic cyanidiophytes in natural habitats can survive under a wide variety of light regimes, and the exploration and elucidation of their long-term photoacclimation mechanisms promises great potential for further biotechnological applications. Ascorbic acid was previously identified as an important protectant against high light stress in Galdieria partita under mixotrophic conditions, yet whether ascorbic acid and its related enzymatic reactive oxygen species (ROS) scavenging system was crucial in photoacclimation for photoautotrophic cyanidiophytes was unclear. The significance of ascorbic acid and related ROS scavenging and antioxidant regenerating enzymes in photoacclimation in the extremophilic red alga Cyanidiococcus yangmingshanensis was investigated by measuring the cellular content of ascorbic acid and the activities of ascorbate-related enzymes. Accumulation of ascorbic acid and activation of the ascorbate-related enzymatic ROS scavenging system characterized the photoacclimation response after cells were transferred from a low light condition at 20 μmol photons m-2 s-1 to various light conditions in the range from 0 to 1000 μmol photons m-2 s-1. The activity of ascorbate peroxidase (APX) was most remarkably enhanced with increasing light intensities and illumination periods among the enzymatic activities being measured. Light-dependent regulation of the APX activity was associated with transcriptional regulation of the chloroplast-targeted APX gene. The important role of the APX activity in photoacclimation was evidenced by the effect of the APX inhibitors on the photosystem II activity and the chlorophyll a content under the high light condition at 1000 μmol photons m-2 s-1. Our findings provide a mechanistic explanation for the acclimation of C. yangmingshanensis to a wide range of light regimes in natural habitats.

Physiological Responses of a Diazotrophic Cyanobacterium to Acidification of Paddy Floodwater: N2 Fixation, Photosynthesis, and Oxidative-Antioxidative Characteristics.

Long-term of excessive fertilization using nitrogen (N) chemical fertilizer caused the acidification of paddy soils. Presently, the impacts of soil acidification on physiological characteristics of diazotrophic cyanobacteria remain unknown. In order to elucidate this issue, the effects of paddy floodwater acidification on activities of respiration, photosynthetic oxygen evolution, and N2 fixation of a paddy diazotrophic cyanobacterium Aliinostoc sp. YYLX235 were investigated in this study. In addition, the origination and quenching of intracellular reactive oxygen species (ROS) were analyzed. The acidification of paddy floodwater decreased intracellular pH and interfered in energy flux from light-harvesting chlorophyll antenna to the reaction center of photosystem II (PS II). Activities of respiration, photosynthetic oxygen evolution, and N2 fixation were decreased by the acidification of paddy floodwater. Accompanied with an increase in ROS, the level of antioxidative system increased. Superoxide dismutase (SOD) and catalase (CAT) were the main enzymatic ROS scavengers in the cells of YYLX235; reduced glutathione (GSH) was the main non-enzymatic antioxidant. Antioxidants and oxidants in the cells of YYLX235 lost balance when the pH of paddy floodwater fell to 5.0 and 4.0, and lipid oxidative damage happened. The results presented in this study suggest that the acidification of paddy soil severely interfered in the photosynthesis of diazotrophic cyanobacteria and induced the production of ROS, which in turn resulted in oxidative damage on diazotrophic cyanobacteria and a decrease in cell vitality.

Effects of 5-aminolevulinic acid on osmotic adjustment and antioxidant system in mung bean under chilling stress

Effects of 5-aminolevulinic acid on osmotic adjustment and antioxidant system in mung bean under chilling stress

Unraveling the Transcriptional Basis of Temperature-Dependent Pinoxaden Resistance in Brachypodium hybridum.

Climate change endangers food security and our ability to feed the ever-increasing human population. Weeds are the most important biotic stress, reducing crop-plant productivity worldwide. Chemical control, the main approach for weed management, can be strongly affected by temperature. Previously, we have shown that temperature-dependent non-target site (NTS) resistance of Brachypodium hybridum is due to enhanced detoxification of acetyl-CoA carboxylase inhibitors. Here, we explored the transcriptional basis of this phenomenon. Plants were characterized for the transcriptional response to herbicide application, high-temperature and their combination, in an attempt to uncover the genetic basis of temperature-dependent pinoxaden resistance. Even though most of the variance among treatments was due to pinoxaden application (61%), plants were able to survive pinoxaden application only when grown under high-temperatures. Biological pathways and expression patterns of members of specific gene families, previously shown to be involved in NTS metabolic resistance to different herbicides, were examined. Cytochrome P450, glucosyl transferase and glutathione-S-transferase genes were found to be up-regulated in response to pinoxaden application under both control and high-temperature conditions. However, biological pathways related to oxidation and glucose conjugation were found to be significantly enriched only under the combination of pinoxaden application and high-temperature. Analysis of reactive oxygen species (ROS) was conducted at several time points after treatment using a probe detecting H2O2/peroxides. Comparison of ROS accumulation among treatments revealed a significant reduction in ROS quantities 24 h after pinoxaden application only under high-temperature conditions. These results may indicate significant activity of enzymatic ROS scavengers that can be correlated with the activation of herbicide-resistance mechanisms. This study shows that up-regulation of genes related to metabolic resistance is not sufficient to explain temperature-dependent pinoxaden resistance. We suggest that elevated activity of enzymatic processes at high-temperature may induce rapid and efficient pinoxaden metabolism leading to temperature-dependent herbicide resistance.

Pre-storage cold acclimation maintained quality of cold-stored cucumber through differentially and orderly activating ROS scavengers

Cucumber fruit are susceptible to chilling injury (CI) during storage and shipping at temperatures below 7–10°C. Pre-storage cold acclimation (PsCA) is a promising postharvest method for reducing CI and maintaining quality, but the reported duration of PsCA varies greatly, partly because no method so far has been developed for evaluating the chilling tolerance induced by PsCA at an early time. Following PsCA at 10°C for 6h, 12h, 24h, 48h and 72h, CI, secondary diseases and relative electrolyte leakage of cucumbers stored at 5°C decreased with PsCA duration, indicating PsCA-induced chilling tolerance in cucumber was positively correlated with the length of exposure to PsCA. Meanwhile, PsCA increased soluble solids and ascorbic acid, and reduced malondialdehyde, O2− and H2O2, suggesting that PsCA improved quality by inhibiting reactive oxygen species (ROS) and maintaining cell membrane integrity. In addition, PsCA enhanced gene expression and activities of four enzymatic ROS scavengers, superoxide dismutase, peroxidase, ascorbate peroxidase, catalase and two non-enzymatic ROS scavengers, AsA and glutathione. However, different ROS scavengers had variable transition points before they were consistently activated, suggesting that ROS scavengers were differentially activated in an orderly way and that the chilling tolerance is positively correlated with the number of activated ROS scavengers, but require enough number of ROS scavengers to be effective. This study provides possibility for early evaluation of PsCA-induced chilling tolerance and for timely arrangement of marketing and processing to reduce losses.

Regulation of hydrogen peroxide accumulation and death of Agrobacterium-transformed cells in tomato transformation

Our previous studies demonstrate that Agrobacterium causes tissue browning that subsequently reduces transformation efficiency in tomato transformation. In addition application of lipoic acid (LA) can reduce tissue browning and increase transformation efficiency in different crops. A major challenge in Agrobacterium-mediated plant transformation is the death of Agrobacterium-transformed cells (DATC), which restricts transgenic plant production. Hydrogen peroxide (H2O2) plays a critical role in oxidative stress. However, little is known about the biochemical and molecular mechanisms for DATC. Our biological and correlation analyses showed that Agrobacterium mediated H2O2 accumulation (HOA) and HOA elevation led to DATC during Agrobacterium-mediated tomato cv. MicroTom transformation. Agrobacterium significantly (P < 0.05) increased 4.2- and 1.4-fold expression of WRKY75 (a H2O2-responsive transcription factor) and superoxide dismutase (SOD), respectively, while the application of 4.4 M H2O2 significantly increased 19- and 2.7-fold expression of WRKY75 and 2-cys peroxiredoxin (Cys), respectively, and decreased fivefold SOD expression, compared with a control. LA application significantly (P < 0.05) reduced 1.6-fold HOA and DATC while it significantly increased 1.7-fold expression of Cys, and reduced 2.2- and 1.4-fold expression of WRKY75 and SOD, respectively. The reduction of HOA and DATC was accompanied by suppression of WRKY75 and SOD and activation of Cys. Our results indicated that DATC was regulated by H2O2 that was triggered by Agrobacterium and LA application through their gene regulation. In addition, HOA was associated with a biotic generating reactive oxygen species (ROS) mechanism, and HOA and DATC were likely regulated by an enzymatic ROS scavenging mechanism during Agrobacterium-mediated tomato transformation.

Comparative Analysis of Defense Responses in Chocolate Spot-Resistant and -Susceptible Faba Bean (Vicia faba) Cultivars Following Infection by the Necrotrophic Fungus Botrytis fabae.

In this study, resistance responses were investigated during the interaction of Botrytis fabae with two faba bean cultivars expressing different levels of resistance against this pathogen, Nubaria (resistant) and Giza 40 (susceptible). Disease severity was assessed on leaves using a rating scale from 1 to 9. Accumulation levels of reactive oxygen species (ROS), lipid peroxidation and antioxidant enzymes (superoxide dismutase, catalase and ascorbate peroxidase) were measured in leaf tissues at different times of infection. The expression profiles of two pathogenesis-related proteins (PRPs) encoded by the genes PR-1 and β-1,3-glucanase were also investigated using reverse transcription RT-PCR analysis. The accumulation of these defense responses was induced significantly in both cultivars upon infection with B. fabae compared with un-inoculated controls. The resistant cultivar showed weaker necrotic symptom expression, less ROS accumulation, a lower rate of lipid peroxidation and higher activity of the enzymatic ROS scavenging system compared with susceptible cultivar. Interestingly, ROS accumulated rapidly in the resistant leaf tissues and peaked during the early stages of infection, whereas accumulation was stronger and more intense in the susceptible tissues in later stages. Moreover, the response of the resistant cultivar to infection was earlier and stronger, exhibiting high transcript accumulation of the PR genes. These results indicated that the induction of oxidant/antioxidant responses and the accumulation of PRPs are part of the faba bean defense mechanism against the necrotrophic fungus B. fabae with a different intensity and timing of induction, depending on the resistance levels.

Effects of Cold Storage Prior to Freezing on Superoxide Dismutase, Glutathione Peroxidase Activities, Level of Total Reactive Oxygen Species and Sperm Quality in Dogs

Reactive oxygen species (ROS) are one of several crucial factors that cause mammalian sperm damage during handling and preservation. Their deleterious effects can be restricted by the action of antioxidants. The present study aimed at investigating: (i) effects of cold storage prior to freezing on activities of enzymatic antioxidants (superoxide dismutase; SOD and glutathione peroxidase; GPx) and level of total reactive oxygen species (tROS); and (ii) effects of SOD or SOD plus GPx supplementation to chilled (3 and 96 h) and frozen-thawed semen. Six privately owned dogs were included. Experiment I: Each pooled semen was divided into three equal aliquots, which were subjected to chilled storage for 3, 24 or 96 h prior to freezing (n = 7). The activities of SOD and GPx in sperm cells and tROS level in chilled and frozen-thawed semen were measured. Experiment II: Pooled semen was divided to be cold stored for 3 or 96 h in three different extenders; (i) Uppsala Equex extender (control), (ii) Uppsala Equex extender supplemented SOD, or (iii) Uppsala Equex extender supplemented with SOD plus GPx. Sperm motility, viability and integrity of acrosome and DNA was evaluated after cold storage and frozen-thawed. The cold storage from 24 h prior to freezing resulted in a decrease in the SOD activity in the frozen-thawed sperm cells whereas the GPx activity and tROS levels were not affected. In addition, the supplementation of SOD plus GPx enhanced the percentage of sperm viability and DNA integrity after cold stored and frozen-thawed. In sum, the SOD activity is compromised by cold storage prior to freezing of dog semen. Addition of GPx is suggested to assist SOD to complete the enzymatic ROS scavenging system in the dog sperm.

Effect of 635 nm Light-Emitting Diode Irradiation on Intracellular Superoxide Anion Scavenging Independent of the Cellular Enzymatic Antioxidant System

The aim of this study was to examine the reactive oxygen species (ROS) that are dissipated by 635 nm irradiation, and the effect of 635 nm irradiation on ROS scavenging system. Intracellular ROS are produced in the form of superoxide anion by either nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase in response to a number of stimuli. Low-level light irradiation decreases the intracellular ROS level and has been used in clinical situations for reducing the level of oxidative stress. Human epithelial cells were exposed to exogenous and endogenous oxidizing agents that promote the generation of harmful ROS. These were then irradiated with 635 nm LED light, 5 mW/cm(2) for 1 h, 18 J/cm(2) or by 470 nm LED light, also 5 mW/cm(2) for 1 h, 18 J/cm(2) on a 9 cm cell culture dish. After irradiation, the MTT reduction method and malondialdehyde (MDA) colorimetric assay were performed in xanthine/xanthine oxidase (XXO)- or hydrogen peroxide (H(2)O(2))-treated HaCaT cells. The superoxide anion was detected by an electron spin resonance (ESR) spectrometer using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap and H(2)O(2) was assayed by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA). Irradiation at 635 nm enhanced cell viability in the XXO-treated HaCaT cells. Also, irradiation had a much lesser effect on cell viability in the HaCaT cells treated with exogenous H(2)O(2) as compared with that in cells treated with N-acetyl-L-cysteine. The level of the superoxide anion increased in response to XXO treatment, and then decreased after 635 nm irradiation. Irradiation with 635 nm led to a decrease in superoxide anion and lipid peroxidation levels in the presence or absence of diethyldithiocarbamate. These results highlight the potential role of 635 nm irradiation in protection against oxidative stress by scavenging superoxide anions. Also, a pathway that is independent of the activities of intracellular enzymatic ROS scavengers, such as superoxide dismutase, glutathione peroxidase and catalase might be involved in its mechanism of action.

Enzymatic reactive oxygen species (ROS) scavenging system in mango varieties resistant and susceptible to malformation

Mango malformation inflicts a serious damage to the mango cultivation, resulting in decreased fruit production as well as quality. A number of biochemical compounds including mangiferin, phenolic compounds, polyphenol oxidase, and phytoalexins have been implicated to play a crucial role in the tolerance/resistance of different mango cultivars to malformation. The present study was attempted to identify a correlation between phytochemicals (phenolic compounds, enzymes, and antioxidants) and the extent of malformation in mango. This study underlines the significance of enzymatic ROS scavenging system in disease resistance/susceptibility. It further advocates strongly that the leaf PPO, LOX and antioxidant enzyme activities can be used as biochemical indicator for screening mango germplasm against malformation disorder. The results of the present investigation establish the role of biochemical metabolites in determining the tolerance of mango varieties to malformation malady which, however, needs to be further explored through the use of molecular tools and markers.

The membrane topological analysis of 3β-hydroxysteroid-Δ24 reductase (DHCR24) on endoplasmic reticulum

DHCR24 encodes 3β-hydroxysteroid-Δ24 reductase, catalyzing the conversion of desmosterol to cholesterol. Our previous study demonstrated that DHCR24 exerts an anti-apoptotic function as a reactive oxygen species (ROS) scavenger, for which it needs its FAD-binding domain. The membrane topology of DHCR24 on endoplasmic reticulum (ER) and the functional significance of its FAD-binding domain are not completely understood. Based on the structure predicted by bioinformatics, we studied the membrane topology of DHCR24 in murine neuroblastoma cells (N2A), using the fluorescent protease protection (FPP) technique. We showed that full-length DHCR24 is localized to the membrane of ER, whereas the predicted transmembrane (TM) domain-deleted DHCR24 mutation is localized to the cytoplasm. The change of DHCR24 localization suggests that the N-terminal TM domain is essential for the ER membrane targeting of DHCR24. The FPP assay demonstrated the membrane topology of DHCR24 with an N-terminal luminal/C-terminal cytoplasmic orientation. Measurement of intracellular ROS using H(2)DCFDA revealed that the ROS levels of cells infected by plasmids driving expression of full-length DHCR24 or the TM domain-deleted DHCR24 mutation after H(2)O(2) exposure were lower than those of control cells, suggesting that the ER membrane targeting of DHCR24 is not required for its enzymatic ROS scavenging activity. Confocal fluorescence microscopy revealed that the DHCR24-overexpressed cells were protected from apoptosis in response to oxidative stress, which was accompanied by a decrease in DHCR24 content on the ER and activation of caspase-3, suggesting that the anti-apoptotic function of DHCR24 is associated with its cleavage by caspase.

The antioxidant glutathione peroxidase family and spermatozoa: A complex story

Oxidative damage is one threat spermatozoa have to face during epididymal maturation and storage. However, it is clear that reactive oxygen species (ROS) are also central for sperm physiology in processes such as sperm maturation and capacitation. It is therefore essential that there exists around sperm cells a fine balance between ROS production and recycling. To do so, sperm cells and epididymal epithelial cells rely on common enzymatic ROS scavengers such as superoxide dismutase (SOD), glutathione peroxidases (GPX) and catalase (CAT) as well as more specific types such as indoleamine dioxygenase (IDO). Among the catalytic triad (SOD/GPX/CAT), the glutathione peroxidase protein family occupies a peculiar position, since several GPX have been found to be present on and around epididymal transiting sperm cells. Here, we will review our present knowledge regarding GPX expression, presence and putative role(s) within the epididymis and on spermatozoa. Taking into account our recent findings regarding the epididymal expression of indoleamine dioxygenase in mouse we will also discuss how we think this superoxide anion recycling enzyme completes the complex ROS generation/recycling balance in this organ.

Oxidative stress as DNA damage in different transgenic tobacco plants

One of the important mechanisms by which plant cells are damaged during adverse environmental conditions is the excessive production of reactive oxygen species (ROS). Major enzymatic ROS scavenging mechanisms of plants include superoxide dismutase (SOD) and catalase (CAT). The aim of this work was to assess leaf cell sensitivity to oxidative stress by measuring DNA damage (Comet assay) in Nicotiana tabacum wild type plants and transgenic lines with deregulated levels of CAT and MnSOD; DNA migration, as DNA damage measure, and nuclei with completely disintegrated head region (hedgehogs), as toxicity parameter, were recorded. A general time dependent increase of DNA migration values was observed within 3 h after hydrogen peroxide treatment, followed by an increase of hedgehog percentage. We showed that hydrogen peroxide treatments trigger different degrees of both DNA migration and hedgehog induction in the transgenic lines. There was only weak difference between the transgenic lines with increased MnSOD levels and wild type plants; but within CAT and MnSOD deficient plants, both levels of DNA migration and hedgehogs were significantly altered.