Glutathione: a key modulator of plant defence and metabolism through multiple mechanisms.

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 an interface between signalling pathways and metabolic reactions that fuel growth and development. It also contributes to the assembly of cell components, biosynthesis of sulphur-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 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.

Impact of high atmospheric carbon dioxide on the biotic stress response of the model cereal species Brachypodium distachyon.

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 (hCO2), on plant immunity in cereals, which provide 60% of human calories. The aim of this study was to determine if hCO2 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 hCO2. 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 hCO2 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 hCO2 and allows identification of pathways that might play a role in this effect.

Thioredoxins m regulate plastid glucose-6-phosphate dehydrogenase activity in Arabidopsis roots under salt stress.

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.

S-Nitrosylation of the histone deacetylase HDA19 stimulates its activity to enhance plant stress tolerance in Arabidopsis.

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.

Quantitative Measurements of Biochemical and Molecular Markers of Oxidative Stress Signaling and Responses.

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.

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.

Quantitative Measurement of Ascorbate and Glutathione by Spectrophotometry.

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.

Measurement of NAD(P)H and NADPH-Generating Enzymes.

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.

Measuring Stress-Induced Changes in Defense Phytohormones and Related Compounds.

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.

Redox Homeostasis and Signaling in a Higher-CO2 World.

Rising CO2 concentrations and their effects on plant productivity present challenging issues. Effects on the photosynthesis/photorespiration balance and changes in primary metabolism are known, caused by the competitive interaction of CO2 and O2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase. However, impacts on stress resistance are less clear. Reactive oxygen species are key players in biotic and abiotic stress responses, but there is no consensus on whether elevated CO2 constitutes a stress. Although high CO2 increases yield in C3 plants, it can also increase cellular oxidation and activate phytohormone defense pathways. Reduction-oxidation processes play key roles in acclimation to high CO2, with specific enzymes acting in compartment-specific signaling. Traditionally, acclimation to high CO2 has been considered in terms of altered carbon gain, but emerging evidence suggests that CO2 is a signal as well as a substrate. Some CO2 effects on defense are likely mediated independently of primary metabolism. Nonetheless, primary photosynthetic metabolism is highly integrated with defense and stress signaling pathways, meaning that plants will be able to acclimate to the changing environment over the coming decades.

Glutathione-dependent denitrosation of GSNOR1 promotes oxidative signalling downstream of H2 O2.

Photorespiratory hydrogen peroxide (H2 O2 ) plays key roles in pathogenesis responses by triggering the salicylic acid (SA) pathway in Arabidopsis. However, factors linking intracellular H2 O2 to activation of the SA pathway remain elusive. In this work, the catalase-deficient Arabidopsis mutant, cat2, was exploited to elucidate the impact of S-nitrosoglutathione reductase 1 (GSNOR1) on H2 O2 -dependent signalling pathways. Introducing the gsnor1-3 mutation into the cat2 background increased S-nitrosothiol levels and abolished cat2-triggered cell death, SA accumulation, and associated gene expression but had little additional effect on the major components of the ascorbate-glutathione system or glycolate oxidase activities. Differential transcriptome profiles between gsnor1-3 and cat2 gsnor1-3 together with damped ROS-triggered gene expression in cat2 gsnor1-3 further indicated that GSNOR1 acts to mediate the SA pathway downstream of H2 O2 . Up-regulation of GSNOR activity was compromised in cat2 cad2 and cat2 pad2 mutants in which glutathione accumulation was genetically prevented. Experiments with purified recombinant GSNOR revealed that the enzyme is posttranslationally regulated by direct denitrosation in a glutathione-dependent manner. Together, our findings identify GSNOR1-controlled nitrosation as a key factor in activation of the SA pathway by H2 O2 and reveal that glutathione is required to maintain this biological function.

PROTEIN PHOSPHATASE 2A-B'γ Controls Botrytis cinerea Resistance and Developmental Leaf Senescence.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

Cytosolic Isocitrate Dehydrogenase from Arabidopsis thaliana Is Regulated by Glutathionylation.

NADP-dependent (Nicotinamide Adénine Dinucléotide 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.

Analyzing the Function of Catalase and the Ascorbate-Glutathione Pathway in H2O2 Processing: Insights from an Experimentally Constrained Kinetic Model.

SIGNIFICANCE: Plant stress involves redox signaling linked to reactive oxygen species such as hydrogen peroxide (H2O2), which can be generated at high rates in photosynthetic cells. The systems that process H2O2 include catalase (CAT) and the ascorbate-glutathione pathway, but interactions between them remain unclear. Modeling can aid interpretation and pinpoint areas for investigation. Recent Advances: Based on emerging data and concepts, we introduce a new experimentally constrained kinetic model to analyze interactions between H2O2, CAT, ascorbate, glutathione, and NADPH. The sensitivity points required for accurate simulation of experimental observations are analyzed, and the implications for H2O2-linked redox signaling are discussed. CRITICAL ISSUES: We discuss several implications of the modeled results, in particular the following. (i) CAT and ascorbate peroxidase can share the load in H2O2 processing even in optimal conditions. (ii) Intracellular H2O2 concentrations more than the low µM range may rarely occur. (iii) Ascorbate redox turnover is largely independent of glutathione until ascorbate peroxidation exceeds a certain value. (iv) NADPH availability may determine glutathione redox status through its influence on monodehydroascorbate reduction. (v) The sensitivity of glutathione status to oxidative stress emphasizes its potential suitability as a sensor of increased H2O2. FUTURE DIRECTIONS: Important future questions include the roles of other antioxidative systems in interacting with CAT and the ascorbate-glutathione pathway as well as the nature and significance of processes that achieve redox exchange between different subcellular compartments. Progress in these areas is likely to be favored by integrating kinetic modeling analyses into experimentally based programs, allowing each approach to inform the other.

Analysis of catalase mutants underscores the essential role of CATALASE2 for plant growth and day length-dependent oxidative signalling.

Three genes encode catalase in Arabidopsis. Although the role of CAT2 in photorespiration is well established, the importance of the different catalases in other processes is less clear. Analysis of cat1, cat2, cat3, cat1 cat2, and cat2 cat3 T-DNA mutants revealed that cat2 had the largest effect on activity in both roots and leaves. Root growth was inhibited in all cat2-containing lines, but this inhibition was prevented by growing plants at high CO2 , suggesting that it is mainly an indirect effect of stress at the leaf level. Analysis of double mutants suggested some overlap between CAT2 and CAT3 functions in leaves and CAT1 and CAT2 in seeds. When plants had been grown to a similar developmental stage in short days or long days, equal-time exposure to oxidative stress caused by genetic or pharmacological inhibition of catalase produced a much stronger induction of H2 O2 marker genes in short day plants. Together, our data (a) underline the importance of CAT2 in basal H2 O2 processing in Arabidopsis; (b) suggest that CAT1 and CAT3 are mainly "backup" or stress-specific enzymes; and (c) establish that day length-dependent responses to catalase deficiency are independent of the duration of oxidative stress.

ROS-related redox regulation and signaling in plants.

As sessile oxygenic organisms with a plastic developmental programme, plants are uniquely positioned to exploit reactive oxygen species (ROS) as powerful signals. Plants harbor numerous ROS-generating pathways, and these oxidants and related redox-active compounds have become tightly embedded into plant function and development during the course of evolution. One dominant view of ROS-removing systems sees them as beneficial antioxidants battling to keep damaging ROS below dangerous levels. However, it is now established that ROS are a necessary part of subcellular and intercellular communication in plants and that some of their signaling functions require ROS-metabolizing systems. For these reasons, it is suggested that "ROS processing systems" would be a more accurate term than "antioxidative systems" to describe cellular components that are most likely to interact with ROS and, in doing so, transmit oxidative signals. Within this framework, our update provides an overview of the complexity and compartmentation of ROS production and removal. We place particular emphasis on the importance of ROS-interacting systems such as the complex cellular thiol network in the redox regulation of phytohormone signaling pathways that are crucial for plant development and defense against external threats.

Glutathione oxidation in response to intracellular H2O2: Key but overlapping roles for dehydroascorbate reductases.

Glutathione is a pivotal molecule in oxidative stress, during which it is potentially oxidized by several pathways linked to H2O2 detoxification. We have investigated the response and functional importance of 3 potential routes for glutathione oxidation pathways mediated by glutathione S-transferases (GST), glutaredoxin-dependent peroxiredoxins (PRXII), and dehydroascorbate reductases (DHAR) in Arabidopsis during oxidative stress. Loss-of-function gstU8, gstU24, gstF8, prxIIE and prxIIF mutants as well as double gstU8 gstU24, gstU8 gstF8, gstU24 gstF8, prxIIE prxIIF mutants were obtained. No mutant lines showed marked changes in their phenotype and glutathione profiles in comparison to the wild-type plants in either optimal conditions or oxidative stress triggered by catalase inhibition. By contrast, multiple loss of DHAR functions markedly decreased glutathione oxidation triggered by catalase deficiency. To assess whether this effect was mediated directly by loss of DHAR enzyme activity, or more indirectly by upregulation of other enzymes involved in glutathione and ascorbate recycling, we measured expression of glutathione reductase (GR) and expression and activity of monodehydroascorbate reductases (MDHAR). No evidence was obtained that either GRs or MDHARs were upregulated in plants lacking DHAR function. Hence, interplay between different DHARs appears to be necessary to couple ascorbate and glutathione pools and to allow glutathione-related signaling during enhanced H2O2 metabolism.

Measurement of Transcripts Associated with Photorespiration and Related Redox Signaling.

To study photorespiration and to characterize related components, gene expression analysis is a central approach. An overview of the experimental setup, protocols, and methods we use to investigate photorespiration-associated gene expression is presented. Within this chapter, we describe simple procedures to experimentally alter the photorespiratory flux and provide protocols for transcriptomic analysis with a focus on genes encoding photorespiratory proteins as well as those induced by photorespiratory hydrogen peroxide (H2O2). Examples of typical results are presented and their significance to understanding redox signaling is discussed.

Climate Change, CO2, and Defense: The Metabolic, Redox, and Signaling Perspectives.

Ongoing human-induced changes in the composition of the atmosphere continue to stimulate interest in the effects of high CO2 on plants, but its potential impact on inducible plant defense pathways remains poorly defined. Recently, several studies have reported that growth at elevated CO2 is sufficient to induce defenses such as the salicylic acid pathway, thereby increasing plant resistance to pathogens. These reports contrast with evidence that defense pathways can be promoted by photorespiration, which is inhibited at high CO2. Here, we review signaling, metabolic, and redox processes modulated by CO2 levels and discuss issues to be resolved in elucidating the relationships between primary metabolism, inducible defense, and biotic stress resistance.