A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: Application to redox profiling during Arabidopsis rosette development

Queval, Guillaume; Noctor, Graham

doi:10.1016/j.ab.2007.01.005

Cited by 392 publications

(280 citation statements)

References 51 publications

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“…In order to compliment the in vivo fluorescent-based assay, we employed a biochemical approach to quantify the various GSH pools in the cells during viral infection. Using this biochemical assay, which is based on the GSH reductase-dependent reduction of 5,5′-dithiobis(2-nitro-benzoic acid) (Ellman's reagent; Tietze, 1969;Queval and Noctor, 2007) (Figures 3b-e), we detected an increase in total GSH (GSH+GSSG, Figure 3b) in cell extracts derived from lytic phase of infection. This result was supported by previous research detecting total GSH increase during infection by means of high-performance liquid chromatography (Gledhill et al, 2012).…”

Section: Expression Profile Of Ros Metabolism Genes During Ehv Infectionmentioning

confidence: 99%

“…Total GSH, GSH, GSSG and GSH/GSSG ratio GSH was measured by the recycling assay, GSH reductase-dependent reduction of 5,5′-dithiobis(2-nitro-benzoic acid) (Ellman's reagent; Tietze, 1969) adjusted for microplate assay (Queval and Noctor, 2007). Briefly, 400 ml cultures were pelleted by mean of centrifugation (17 000 g, 10 min, 4°C) plunged in to liquid nitrogen and were kept in − 80°C.…”

Section: Detection Of Intracellular Rosmentioning

confidence: 99%

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Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean

et al. 2016

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The cosmopolitan coccolithophore Emiliania huxleyi is a unicellular eukaryotic alga responsible for vast blooms in the ocean. These blooms have immense impact on large biogeochemical cycles and are terminated by a specific large double-stranded DNA E. huxleyi virus (EhV, Phycodnaviridae). EhV infection is accompanied by induction of hallmarks of programmed cell death and production of reactive oxygen species (ROS). Here we characterized alterations in ROS metabolism and explored its role during infection. Transcriptomic analysis of ROS-related genes predicted an increase in glutathione (GSH) and H 2 O 2 production during infection. In accordance, using biochemical assays and specific fluorescent probes we demonstrated the overproduction of GSH during lytic infection. We also showed that H 2 O 2 production, rather than superoxide, is the predominant ROS during the onset of the lytic phase of infection. Using flow cytometry, confocal microscopy and multispectral imaging flow cytometry, we showed that the profound co-production of H 2 O 2 and GSH occurred in the same subpopulation of cells but at different subcellular localization. Positively stained cells for GSH and H 2 O 2 were highly infected compared with negatively stained cells. Inhibition of ROS production by application of a peroxidase inhibitor or an H 2 O 2 scavenger inhibited host cell death and reduced viral production. We conclude that viral infection induced remodeling of the host antioxidant network that is essential for a successful viral replication cycle. This study provides insight into viral replication strategy and suggests the use of specific cellular markers to identify and quantify the extent of active viral infection during E. huxleyi blooms in the ocean.

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Section: Expression Profile Of Ros Metabolism Genes During Ehv Infectionmentioning

confidence: 99%

Section: Detection Of Intracellular Rosmentioning

confidence: 99%

Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean

et al. 2016

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“…Briefly, catalase was measured by the removal of H 2 O 2 monitored at 240 nm, DHAR as GSH-dependent formation of ascorbate from DHA at 265 nm, APX as H 2 O 2 -dependent ascorbate oxidation at 290 nm, and GR as GSSG-dependent NADPH oxidation at 340 nm. Oxidized and reduced forms of glutathione and ascorbate were measured by plate-reader assay as described by Queval and Noctor (2007). Total SA was measured according to the protocol of Chaouch et al (2010).…”

Section: Enzyme Assays and Metabolite Analysismentioning

confidence: 99%

Cytosolic and Chloroplastic DHARs Cooperate in Oxidative Stress-Driven Activation of the Salicylic Acid Pathway

Rahantaniaina

Li²,

Châtel-Innocenti³

et al. 2017

Plant Physiol.

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The complexity of plant antioxidative systems gives rise to many unresolved questions. One relates to the functional importance of dehydroascorbate reductases (DHARs) in interactions between ascorbate and glutathione. To investigate this issue, we produced a complete set of loss-of-function mutants for the three annotated Arabidopsis (Arabidopsis thaliana) DHARs. The combined loss of DHAR1 and DHAR3 expression decreased extractable activity to very low levels but had little effect on phenotype or ascorbate and glutathione pools in standard conditions. An analysis of the subcellular localization of the DHARs in Arabidopsis lines stably transformed with GFP fusion proteins revealed that DHAR1 and DHAR2 are cytosolic while DHAR3 is chloroplastic, with no evidence for peroxisomal or mitochondrial localizations. When the mutations were introduced into an oxidative stress genetic background (cat2), the dhar1 dhar2 combination decreased glutathione oxidation and inhibited cat2-triggered induction of the salicylic acid pathway. These effects were reversed in cat2 dhar1 dhar2 dhar3 complemented with any of the three DHARs. The data suggest that (1) DHAR can be decreased to negligible levels without marked effects on ascorbate pools, (2) the cytosolic isoforms are particularly important in coupling intracellular hydrogen peroxide metabolism to glutathione oxidation, and (3) DHAR-dependent glutathione oxidation influences redox-driven salicylic acid accumulation.

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“…Samples were centrifuged at 13,000 rpm for 10 min at 6°C, and then supernatant pH was adjusted to between 5 and 6. Glutathione levels were assayed in samples and standards using a platereader protocol, as described in detail by Queval and Noctor (2007). Briefly, the rate of 5,59-dithiobis(2-nitrobenzoic acid) reduction to thionitrobenzoic acid by GSH was measured by following the increase in A 412 in a system containing glutathione reductase to recycle GSSG.…”

Section: Glutathione Measurementsmentioning

confidence: 99%

Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene

et al. 2014

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The explosive 2,4,6-trinitrotoluene (TNT) is a major worldwide military pollutant. The presence of this toxic and highly persistent pollutant, particularly at military sites and former manufacturing facilities, presents various health and environmental concerns. Due to the chemically resistant structure of TNT, it has proven to be highly recalcitrant to biodegradation in the environment. Here, we demonstrate the importance of two glutathione transferases (GSTs), GST-U24 and GST-U25, from Arabidopsis (Arabidopsis thaliana) that are specifically up-regulated in response to TNT exposure. To assess the role of GST-U24 and GST-U25, we purified and characterized recombinant forms of both enzymes and demonstrated the formation of three TNT glutathionyl products. Importantly, GST-U25 catalyzed the denitration of TNT to form 2-glutathionyl-4,6-dinitrotoluene, a product that is likely to be more amenable to subsequent biodegradation in the environment. Despite the presence of this biochemical detoxification pathway in plants, physiological concentrations of GST-U24 and GST-U25 result in only a limited innate ability to cope with the levels of TNT found at contaminated sites. We demonstrate that Arabidopsis plants overexpressing GST-U24 and GST-U25 exhibit significantly enhanced ability to withstand and detoxify TNT, properties that could be applied for in planta detoxification of TNT in the field. The overexpressing lines removed significantly more TNT from soil and exhibited a corresponding reduction in glutathione levels when compared with wild-type plants. However, in the absence of TNT, overexpression of these GSTs reduces root and shoot biomass, and although glutathione levels are not affected, this effect has implications for xenobiotic detoxification.The containment and cleanup of environmental pollutants is increasingly both a legal requirement and a responsible action in many developed countries. The most commonly used explosives in military weapons are 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and their continual use, along with production and decommissioning, are progressively contaminating millions of hectares of military land . Bioremediation of TNT is particularly challenging, as the electron-withdrawing properties of the three nitro groups render the aromatic ring particularly resistant to oxidative attack and ring cleavage by microbial oxygenases, which in the environment are normally central to the biodegradation of aromatic compounds (Qasim et al., 2007). In the United States, the Environmental Protection Agency and the military are addressing methods by which toxic TNT and RDX can be contained and detoxified on active military training ranges. One way this problem might be tackled is through the use of plants that are adapted to detoxify these compounds. This could be achieved either by traditional breeding programs or by genetic modification, as has been demonstrated previously for both RDX and TNT (Hannink et al., 2001;Rylott et al., 2006;Jackson et al., 2007).In t...

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A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: Application to redox profiling during Arabidopsis rosette development

Cited by 392 publications

References 51 publications

Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean

Modulation of host ROS metabolism is essential for viral infection of a bloom-forming coccolithophore in the ocean

Cytosolic and Chloroplastic DHARs Cooperate in Oxidative Stress-Driven Activation of the Salicylic Acid Pathway

Arabidopsis Glutathione Transferases U24 and U25 Exhibit a Range of Detoxification Activities with the Environmental Pollutant and Explosive, 2,4,6-Trinitrotoluene

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