Abstract The oxidative pentose phosphate pathway (OPPP) is a source of cellular NADPH, generated through the sequential activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH). Using the catalase-deficient cat2 background as a model for H 2 O 2 -triggered salicylic acid (SA) signaling we identified the cytosolic G6PDH isoform G6PD5 as a key determinant of redox homeostasis and SA-dependent defense activation (Trémulot et al., companion manuscript). However, the mechanisms underlying this function remain enigmatic. In this work, genetic and transcriptomic analyses show that the role of G6PD5 cannot be explained solely by altered NADPH generation for either NADPH oxidases or the ascorbate-glutathione pathway, suggesting other possible links. To identify such links, a forward genetic screen was employed. We searched for mutations that modulate the suppressed lesion phenotype in cat2 g6pd5 in a photorespiration-dependent manner. This screen identified a mutation in PGD2 , encoding the cytosolic 6PGDH. Strikingly, functional analyses of mutants and overexpression lines revealed that PGD2 exerts effects opposite to those of G6PD5 in SA signaling. Our observations uncover an unexpected antagonism between the two cytosolic NADPH-producing steps within the OPPP. Pharmacological analyses support a signaling role for the metabolic intermediate 6-phosphogluconolactone in linking the OPPP to SA signaling. These findings indicate that the OPPP is not solely a source of reducing power during oxidative stress but also acts as a signaling module in which metabolic intermediates contribute to the control of stress-induced immune responses.

Accumulating evidence shows that reversible protein S-nitrosylation is essential for H2O2 homoeostasis and signalling. However, roles for denitrosylation in such oxidative signalling remain poorly understood. Here, we examined this question using the Arabidopsis catalase-defective mutant, cat2, in which oxidative stress induces both glutathione accumulation and salicylic acid (SA) pathways. Induction of these pathways was accompanied by enhanced thioredoxin (TRXH5) expression, and oxidative stress-induced activation of the SA pathway was compromised when TRXH5 expression was genetically disabled, whereas TRXH5 overexpression stimulates H2O2-triggered SA responses. Intriguingly, TRXh5-reinforced SA responses were antagonised by glutathione (GSH) deficiency when introducing additional pad2 mutation, localised in the GLUTAMATE-CYSTEINE LIGASE gene encoding the first enzyme of glutathione biosynthesis. Further analysis revealed that the two active cysteine residues of recombinant TRXh5 can be denitrosylated by GSH. Blocking glutathione accumulation increased more TRXh5-SNO formation in TRXH5-YFP cat2 pad2 trxh5 than in TRXH5-YFP cat2 trxh5. Furthermore, S-nitrosoglutathione reductase (GSNOR) was capable of physically interacting with TRXh5, and was also required for GSH-dependent TRXh5 denitrosylation and TRXh5-enhanced SA responses during oxidative stress. Collectively, these data suggest that GSH/GSNOR constitutes an active denitrosylating module that works together with the canonical NADPH-dependent TRX-reducing pathway to sustain cytosolic TRXh5 operation within the oxidative signalling framework.

Abstract Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the oxidative pentose phosphate pathway, generating NADPH to sustain redox metabolism and signaling. However, whether individual G6PDH isoforms directly regulate oxidative stress signaling remains unclear. To determine the contribution of the different Arabidopsis G6PDH isoforms to oxidative stress signaling, we introduced single T-DNA mutants into the catalase-deficient cat2 background, a genetic system in which intracellular H 2 O 2 production activates salicylic acid (SA)-dependent cell death and defense pathways. Interestingly, impairment of cytosolic, but not chloroplastic G6PDH activity suppressed cat2 -triggered phenotypes, with loss of G6PD5 function fully abolishing lesion formation. The cat2 g6pd5 double mutant phenocopied the SA biosynthesis-deficient mutant cat2 sid2 and showed reversion of defense responses as well as metabolomic and transcriptomic profiles to the wild-type state. Strikingly, despite the suppression of SA-dependent lesions, loss of G6PD5 activity does not appear to reduce stress intensity. On the contrary, cat2 g6pd5 plants exhibit increased glutathione synthesis and oxidation, elevated expression of oxidative stress marker genes, and enhanced accumulation of reactive nitrogen species relative to cat2 . Protein-protein interaction analyses revealed that G6PD5 associates with several redox and defense-related proteins. In particular, we confirmed a physical interaction between G6PD5 and thioredoxin h5, a key component of redox-dependent SA signaling. However, analysis of cat2 trxh5 and cat2 npr1 lines indicated that this interaction alone cannot explain the G6PD5-dependent control of SA responses. Our work reveals that cytosolic G6PD5 integrates redox metabolism with immune signaling to control plant responses to oxidative stress.

Plant viruses cause significant crop losses, a situation that could worsen due to anthropogenic activities driving global climate change, one factor of which is the increase in atmospheric CO2 concentration. This study assessed the impact of elevated CO2 concentration (eCO2, 1000 vs. 400 ppm) on two genotypes of common bean (Phaseolus vulgaris L.), one susceptible and one resistant, infected with bean pod mottle virus (BPMV, Comovirus siliquae). For both genotypes, we found that plant growth, development and physiology were not enhanced under eCO2 enrichment in healthy plants, at the stage of BPMV inoculation. Under eCO2, the number of primary infection sites was reduced in both genotypes. Consistently, viral titre in inoculated leaves was lower, suggesting an enhanced resistance to BPMV in both genotypes under eCO2. To investigate the underlying mechanisms, we studied the expression of genes involved in different antiviral immune pathways: salicylic acid (SA)-signalling, RNA silencing and PAMP-triggered immunity (PTI) pathways. Under our experimental conditions, eCO2 neither primed the SA-signalling pathway nor the PTI pathway, in both genotypes. However, eCO2 seems to prime the RNA silencing pathway in the resistant genotype, and to a lesser extent, in the susceptible genotype.

Ascorbate and glutathione are water-soluble compounds that are found at high concentrations in many plant tissues. A close association between the two molecules has been noted almost since the time Planta was founded, 100 years ago. Although both have many functions, much attention has been paid to their influence as antioxidants. One of the conceptual turning-points regarding the significance of these compounds in plants occurred in the second half of the 1970s, when the ascorbate–glutathione pathway was first characterized as a chloroplastic antioxidative process. Now known as the Foyer–Halliwell–Asada pathway, this sequence of reactions notably links reduction of H2O2, catalysed by ascorbate peroxidase, to oxidation of NADH or NADPH, catalysed by monodehydroascorbate reductase and glutathione reductase. One of the papers that laid the foundation stones of the pathway was Foyer and Halliwell (Planta 133:21–25, 1976). This perspective takes a look back at the contributions of this and related work in the context of plant biology research at the time, and considers the importance of the pathway within our current understanding of reactive oxygen species biology and redox homeostasis and signalling. Emphasis is placed on the advances in our knowledge of the genes and proteins involved and the potential metabolic flexibility of the pathway, as well as its place within the highly intricate plant network of H2O2-metabolising systems.

The antioxidative enzyme monodehydroascorbate reductase (MDHAR) is represented by five genes in Arabidopsis, including four that encode cytosolic and peroxisomal proteins. The in planta importance of these specific isoforms during oxidative stress remain to be characterised. T-DNA mutants for MDAR genes encoding cytosolic and peroxisomal isoforms were studied. To examine their roles in conditions of intracellular oxidative stress, mutants were crossed with a cat2 line lacking the major leaf catalase. Enzyme assays in mdar mutants and of recombinant MDHARs suggest that peroxisomal MDHAR1 and cytosolic MDHAR2 are major players in leaf NADH- and NADPH-dependent activities, respectively. All mutants showed a wild-type phenotype when grown in standard conditions. In the cat2 background, loss of peroxisomal MDHAR functions decreased growth whereas loss of the cytosolic MDHAR2 function had no effect on growth but annulled a large part of transcriptomic and phenotypic responses to oxidative stress. The effects of the mdar2 mutation included decreased salicylic acid accumulation and enhanced glutathione oxidation, and were reverted by complementation with the MDAR2 sequence. Together, the data show that the cytosolic MDHAR2 is dispensable in optimal conditions but essential to promote biotic defence responses triggered by oxidative stress.

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Redox reactions are fundamental to energy conversion in living cells, and also determine and tune responses to the environment. Within this context, the tripeptide glutathione plays numerous roles. As an important antioxidant, glutathione confers redox stability on the cell and also acts as an interface between signalling pathways and metabolic reactions that fuel growth and development. It also contributes to the assembly of cell components, biosynthesis of sulfur-containing metabolites, inactivation of potentially deleterious compounds, and control of hormonal signalling intensity. The multiplicity of these roles probably explains why glutathione status has been implicated in influencing plant responses to many different conditions. In particular, there is now a considerable body of evidence showing that glutathione is a crucial player in governing the outcome of biotic stresses. This review provides an overview of glutathione synthesis, transport, degradation, and redox turnover in plants. It examines the expression of genes associated with these processes during pathogen challenge and related conditions, and considers the diversity of mechanisms by which glutathione can influence protein function and gene expression.

Plants contain several NADPH-producing enzymes including glucose-6-phosphate dehydrogenases (G6PDH) with different sub-cellular localizations. The activity of plastidial G6PDHs is redox-regulated by thioredoxins (TRX). Although specific TRXs are known to regulate chloroplastic isoforms of G6PDH, little information is available for plastidic isoforms found in heterotrophic organs or tissues. Here, we investigated TRX regulation of the two G6PDH plastidic isoforms of Arabidopsis roots during exposure to a mild salt stress. We report that in vitro m-type TRXs are the most efficient regulators of the G6PDH2 and G6PDH3 mainly found in Arabidopsis roots. While expression of the corresponding G6PD and plastidic TRX genes was marginally affected by salt, it impaired root growth of several of the corresponding mutant lines. Using an in situ assay for G6PDH, G6PDH2 was found to be the major contributor to salt-induced increases in activity, while data from ROS assays further provide in vivo evidence that TRX m acts in redox regulation during salt stress. Taken together, our data suggest that regulation of plastid G6PDH activity by TRX m may be an important player regulating NADPH production in Arabidopsis roots undergoing salt stress.

Studies of the Arabidopsis cat2 mutant lacking the major leaf isoform of catalase have allowed the potential impact of intracellular H2O2 on plant function to be studied. Here, we report a robust analysis of modified gene expression associated with key families involved in metabolite modification in cat2. Through a combined transcriptomic and metabolomic analysis focused on the salicylic acid (SA) and jasmonic acid (JA) pathways, we report key features of the metabolic signatures linked to oxidative stress-induced signaling via these defence hormones and discuss the enzymes that are likely to be involved in determining these features. We provide evidence that specific UDP-glycosyl transferases contribute to the glucosylation of SA that accumulates as a result of oxidative stress in cat2. Glycosides of dihydroxybenzoic acids that accumulate alongside SA in cat2 are identified and, based on the expression of candidate genes, likely routes for their production are discussed. We also report that enhanced intracellular H2O2 triggers induction of genes encoding different enzymes that can metabolize JA. Integrated analysis of metabolite and transcript profiles suggests that a gene network involving specific hydrolases, hydroxylases, and sulfotransferases functions to limit accumulation of the most active jasmonates during oxidative stress.

Arabidopsis histone deacetylase HDA19 is required for gene expression programs of a large spectrum of plant developmental and stress-responsive pathways. How this enzyme senses cellular environment to control its activity remains unclear. In this work, we show that HDA19 is post-translationally modified by S-nitrosylation at 4 Cysteine (Cys) residues. HDA19 S-nitrosylation depends on the cellular nitric oxide level, which is enhanced under oxidative stress. We find that HDA19 is required for cellular redox homeostasis and plant tolerance to oxidative stress, which in turn stimulates its nuclear enrichment, S-nitrosylation and epigenetic functions including binding to genomic targets, histone deacetylation and gene repression. The Cys137 of the protein is involved in basal and stress-induced S-nitrosylation, and is required for HDA19 functions in developmental, stress-responsive and epigenetic controls. Together, these results indicate that S-nitrosylation regulates HDA19 activity and is a mechanism of redox-sensing for chromatin regulation of plant tolerance to stress.

Losses due to disease and climate change are among the most important issues currently facing crop production. It is therefore important to establish the impact of climate change, and particularly of high carbon dioxide (hCO(2)), on plant immunity in cereals, which provide 60% of human calories. The aim of this study was to determine if hCO(2) impacts Brachypodium distachyon immunity, a model plant for temperate cereals. Plants were grown in air (430 ppm CO2) and at two high CO2 conditions, one that is relevant to projections within the coming century (1000 ppm) and a concentration sufficient to saturate photosynthesis (3000 ppm). The following measurements were performed: phenotyping and growth, salicylic acid contents, pathogen resistance tests, and RNAseq analysis of the transcriptome. Improved shoot development was observed at both 1000 and 3000 ppm. A transcriptomic analysis pointed to an increase in primary metabolism capacity under hCO(2). Alongside this effect, up-regulation of genes associated with secondary metabolism was also observed. This effect was especially evident for the terpenoid and phenylpropanoid pathways, and was accompanied by enhanced expression of immunity-related genes and accumulation of salicylic acid. Pathogen tests using the fungus Magnaporthe oryzae revealed that hCO(2) had a complex effect, with enhanced susceptibility to infection but no increase in fungal development. The study reveals that immunity in B. distachyon is modulated by growth at hCO(2) and allows identification of pathways that might play a role in this effect.

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.

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.

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 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.

1 Ludwig Maximilians University of Munich, Faculty of Biology, LMU Biocenter, Grosshaderner Str. 2-4, 82152 Planegg-Martinsried, Germany 2 Biosciences, College of Life and Environmental Sciences, University of Exeter,Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK 3 Department of Plant Biotechnology and Bioinformatics, Ghent University, Center for Plant Systems Biology, VIB, Technologiepark-Zwijnaarde 71, 9052 Ghent, Belgium 4 University of Bielefeld, Faculty of Biology, Biochemistry and Physiology of Plants, 33615 Bielefeld, Germany 5 Institut Universitaire de France (IUF), Université Paris-Saclay, CNRS, INRAE, Univ Evry, Institute of Plant Sciences Paris-Saclay (IPS2), 91405, Orsay, France

Arabidopsis histone deacetylase HDA19 is required for gene expression programs of a large spectrum of plant developmental and stress-responsive pathways. How this enzyme senses cellular environment to control its activity remains unclear. In this work, we show that HDA19 is post-translationally modified by S-nitrosylation at 4 Cysteine (Cys) residues. HDA19 S-nitrosylation depends on cellular nitric oxide (NO) levels and is enhanced under stress. We find that HDA19 is required for cellular redox homeostasis and plant tolerance to oxidative stress which in turn stimulates its nuclear enrichment and S-nitrosylation and epigenetic functions including binding to genomic targets, histone deacetylation, and gene repression. Cys137 is a major site for both NO-dependent and stress-enhanced S-nitrosylation of the protein and is required for HDA19 functions in developmental, stress-responsive, and epigenetic controls. Finally, we show that the nucleocytosolic glutaredoxin GRXS17 that functions as a redox-related holdase interacts with HDA19 and is indispensable for HDA19 S-nitrosylation. Together, these results indicate that S-nitrosylation regulates HDA19 activity and is a mechanism of redox-sensing for chromatin regulation of plant tolerance to stress.