Titel Deelnemers "Korte inhoud" "Quantitative measurements of biochemical and molecular markers of oxidative stress signaling and responses" "Graham Noctor, Mathias Cohen, Frank Van Breusegem, Amna M'Hamdi" "Increases in cellular oxidation are a part of most plant responses to challenging conditions and are commonly described as oxidative stress. While this phenomenon is closely related to the accumulation of reactive oxygen species, these latter compounds can be difficult to measure. Complementary measurements to assess cellular redox state are, therefore, very useful in studies of plant responses to stress. Here, we detail protocols for three complementary approaches that can be used to assess the intensity of oxidative stress. These involve quantification of marker transcripts, assays of the extractable activities of major antioxidative enzymes, and measurement of antioxidant buffers. We confirm experimentally that the data obtained by such approaches can provide reliable information on the intensity of oxidative stress." "Metabolite modification in oxidative stress responses : a case study of two defense hormones" "Caroline Lelarge-Trouverie, Mathias Cohen, Lug Trémulot, Frank Van Breusegem, Amna M'Hamdi, Graham Noctor" "Hydrogen peroxide in plants" "Amna M'Hamdi" "Quantitative measurement of ascorbate and glutathione by spectrophotometry" "Graham Noctor, Amna M'Hamdi" "Ascorbate and glutathione are key chemical antioxidants present at relatively high concentrations in plant cells. They are also reducing cofactors for enzymes that process hydrogen peroxide in the ascorbate-glutathione pathway. Due to these two related biochemical functions, the compounds form an interface between reactive oxygen species and sensitive cellular components. Therefore, their status can provide reliable and direct information on cell redox state, signaling, and plant health. While several methods exist for quantification of ascorbate and glutathione, simple enzyme-dependent assays allow them to be measured easily and inexpensively in common extracts. This chapter describes a protocol to measure total contents, as well as the major oxidized and reduced forms, of both compounds in plant tissues." "Measuring stress-induced changes in defense phytohormones and related compounds" "Caroline Lelarge-Trouverie, Amna M'Hamdi, Florence Guérard, Graham Noctor" "Measuring quantitative changes in plant hormones and derivatives is crucial to understand how reactive oxygen species trigger signaling cascades to regulate stress responses. In this chapter, we describe the liquid chromatography-mass spectrometry procedure that we use to extract and quantify salicylic acid (SA), jasmonic acid (JA), and related compounds in common extracts of Arabidopsis tissue. The method can provide quantitative data on SA, SA glucosides, and JA, as well as information on oxidized and conjugated forms of these compounds and related derivatives of benzoic acid." "Measurement of NAD(P)H and NADPH-generating enzymes" "Amna M'Hamdi, Frank Van Breusegem, Graham Noctor" "Pyridine nucleotides (NAD(H) and NADP(H)) are key redox carriers in cells and may also have other functions related to stress. These two molecules are crucial in linking metabolism to electron transport chains in photosynthesis and respiration, but they are also critical for ensuring redox signaling and homeostasis during episodes of stress. This is especially the case for NADPH, which must be generated from its oxidized form, NADP+, by key dehydrogenases. Here, we describe methods that can be used to assay contents and redox states of NAD(H) and NADP(H), as well as simple assays to measure the capacity of two key NADPH-generating enzymes." "EAL4 methyltransferase plays a specific role in response to oxidative stress" "Amna M'Hamdi, Huaming He, Frank Van Breusegem" "Cytosolic isocitrate dehydrogenase from Arabidopsis thaliana is regulated by glutathionylation" "Adnan Khan Niazi, Laetitia Bariat, Christophe Riondet, Christine Carapito, Amna M'Hamdi, Graham Noctor, Jean-Philippe Reichheld" "NADP-dependent (Nicotinamide Adenine Dinucleotide Phosphate-dependent) isocitrate dehydrogenases (NADP-ICDH) are metabolic enzymes involved in 2-oxoglutarate biosynthesis, but they also supply cells with NADPH. Different NADP-ICDH genes are found in Arabidopsis among which a single gene encodes for a cytosolic ICDH (cICDH) isoform. Here, we show that cICDH is susceptible to oxidation and that several cysteine (Cys) residues are prone to S-nitrosylation upon nitrosoglutathione (GSNO) treatment. Moreover, we identified a single S-glutathionylated cysteine Cys363 by mass-spectrometry analyses. Modeling analyses suggest that Cys363 is not located in the close proximity of the cICDH active site. In addition, mutation of Cys363 consistently does not modify the activity of cICDH. However, it does affect the sensitivity of the enzyme to GSNO, indicating that S-glutathionylation of Cys363 is involved in the inhibition of cICDH activity upon GSNO treatments. We also show that glutaredoxin are able to rescue the GSNO-dependent inhibition of cICDH activity, suggesting that they act as a deglutathionylation system in vitro. The glutaredoxin system, conversely to the thioredoxin system, is able to remove S-nitrosothiol adducts from cICDH. Finally, NADP-ICDH activities were decreased both in a catalase2 mutant and in mutants affected in thiol reduction systems, suggesting a role of the thiol reduction systems to protect NADP-ICDH activities in planta. In line with our observations in Arabidopsis, we found that the human recombinant NADP-ICDH activity is also sensitive to oxidation in vitro, suggesting that this redox mechanism might be shared by other ICDH isoforms." "Measurement of transcripts associated with photorespiration and related redox signaling" "Amna M'Hamdi, Pavel Kerchev, Graham Noctor, Frank Van Breusegem" "The ROS wheel: refining ROS transcriptional footprints" "Amna M'Hamdi, Simon Stael, Veronique Storme, Pavel Kerchev, Graham Noctor, Frank Van Breusegem"