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.

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.

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.

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.

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

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.

Viewing oxidative stress through the lens of oxidative signalling rather than damage.

Concepts of the roles of reactive oxygen species (ROS) in plants and animals have shifted in recent years from focusing on oxidative damage effects to the current view of ROS as universal signalling metabolites. Rather than having two opposing activities, i.e. damage and signalling, the emerging concept is that all types of oxidative modification/damage are involved in signalling, not least in the induction of repair processes. Examining the multifaceted roles of ROS as crucial cellular signals, we highlight as an example the loss of photosystem II function called photoinhibition, where photoprotection has classically been conflated with oxidative damage.

High CO2 Primes Plant Biotic Stress Defences through Redox-Linked Pathways.

Industrial activities have caused tropospheric CO2 concentrations to increase over the last two centuries, a trend that is predicted to continue for at least the next several decades. Here, we report that growth of plants in a CO2-enriched environment activates responses that are central to defense against pathogenic attack. Salicylic acid accumulation was triggered by high-growth CO2 in Arabidopsis (Arabidopsis thaliana) and other plants such as bean (Phaseolus vulgaris). A detailed analysis in Arabidopsis revealed that elevated CO2 primes multiple defense pathways, leading to increased resistance to bacterial and fungal challenge. Analysis of gene-specific mutants provided no evidence that activation of plant defense pathways by high CO2 was caused by stomatal closure. Rather, the activation is partly linked to metabolic effects involving redox signaling. In support of this, genetic modification of redox components (glutathione contents and NADPH-generating enzymes) prevents full priming of the salicylic acid pathway and associated resistance by high CO2 The data point to a particularly influential role for the nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase, a cytosolic enzyme whose role in plants remains unclear. Our observations add new information on relationships between high CO2 and oxidative signaling and provide novel insight into plant stress responses in conditions of increased CO2.

The secondary metabolism glycosyltransferases UGT73B3 and UGT73B5 are components of redox status in resistance of Arabidopsis to Pseudomonas syringae pv. tomato.

Secondary metabolism plant glycosyltransferases (UGTs) ensure conjugation of sugar moieties to secondary metabolites (SMs) and glycosylation contributes to the great diversity, reactivity and regulation of SMs. UGT73B3 and UGT73B5, two UGTs of Arabidopsis thaliana (Arabidopsis), are involved in the hypersensitive response (HR) to the avirulent bacteria Pseudomonas syringae pv. tomato (Pst-AvrRpm1), but their function in planta is unknown. Here, we report that ugt73b3, ugt73b5 and ugt73b3 ugt73b5 T-DNA insertion mutants exhibited an accumulation of reactive oxygen species (ROS), an enhanced cell death during the HR to Pst-AvrRpm1, whereas glutathione levels increased in the single mutants. In silico analyses indicate that UGT73B3 and UGT73B5 belong to the early salicylic acid (SA)-induced genes whose pathogen-induced expression is co-regulated with genes related to cellular redox homeostasis and general detoxification. Analyses of metabolic alterations in ugt mutants reveal modification of SA and scopoletin contents which correlate with redox perturbation, and indicate quantitative modifications in the pattern of tryptophan-derived SM accumulation after Pst-AvrRpm1 inoculation. Our data suggest that UGT73B3 and UGT73B5 participate in regulation of redox status and general detoxification of ROS-reactive SMs during the HR to Pst-AvrRpm1, and that decreased resistance to Pst-AvrRpm1 in ugt mutants is tightly linked to redox perturbation.

Glutathione-dependent phytohormone responses: teasing apart signaling and antioxidant functions.

Cellular redox state is regulated by numerous components. The thiol-disulfide compound, glutathione, is considered to be one of the most significant, owing to its antioxidant power and potential influence over protein structure and function. While signaling roles for glutathione in plants have been suggested for several years, hard proof is scarce. Recently, through an approach based on genetic manipulation of glutathione in an oxidative stress background, we reported evidence that glutathione status is important to allow intracellular oxidation to activate pathogenesis-related phytohormone signaling pathways. This effect does not seem to be caused by changes in glutathione antioxidant capacity, and appears to be distinct to regulation through known players in pathogenesis responses, such as NPR1. Our data therefore suggest that new glutathione-dependent components that link oxidative stress to response outputs await discovery.

Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod-influenced responses to ozone using Arabidopsis knockout mutants.

Oxidative stress caused by ozone (O3 ) affects plant development, but the roles of specific redox-homeostatic enzymes in O3 responses are still unclear. While growth day length may affect oxidative stress outcomes, the potential influence of day length context on equal-time exposures to O3 is not known. In Arabidopsis Col-0, day length affected the outcome of O3 exposure. In short-days (SD), few lesions were elicited by treatments that caused extensive lesions in long days (LD). Lesion formation was not associated with significant perturbation of glutathione, ascorbate, NADP(H) or NAD(H). To investigate roles of two genes potentially underpinning this redox stability, O3 responses of mutants for cytosolic NADP-isocitrate dehydrogenase (icdh) and glutathione reductase 1 (gr1) were analysed. Loss of ICDH function did not affect O3 -induced lesions, but slightly increased glutathione oxidation, induction of other cytosolic NADPH-producing enzymes and pathogenesis-related gene 1 (PR1). In gr1, O3 -triggered lesions, salicylic acid accumulation, and induction of PR1 were all decreased relative to Col-0 despite enhanced accumulation of glutathione. Thus, even at identical irradiance and equal-time exposures, day length strongly influences phenotypes triggered by oxidants of atmospheric origin, while in addition to its antioxidant function, the GR-glutathione system seems to play novel signalling roles during O3 exposure.

Functional analysis of Arabidopsis mutants points to novel roles for glutathione in coupling H(2)O(2) to activation of salicylic acid accumulation and signaling.

AIMS: Through its interaction with H(2)O(2), glutathione is a candidate for transmission of signals in plant responses to pathogens, but identification of signaling roles is complicated by its antioxidant function. Using a genetic approach based on a conditional catalase-deficient Arabidopsis mutant, cat2, this study aimed at establishing whether GSH plays an important functional role in the transmission of signals downstream of H(2)O(2). RESULTS: Introducing the cad2 or allelic mutations in the glutathione synthesis pathway into cat2 blocked H(2)O(2)-triggered GSH oxidation and accumulation. While no effects on NADP(H) or ascorbate were observed, and H(2)O(2)-induced decreases in growth were maintained, blocking GSH modulation antagonized salicylic acid (SA) accumulation and SA-dependent responses. Other novel double and triple mutants were produced and compared with cat2 cad2 at the levels of phenotype, expression of marker genes, nontargeted metabolite profiling, accumulation of SA, and bacterial resistance. Most of the effects of the cad2 mutation on H(2)O(2)-triggered responses were distinct from those produced by mutations for GLUTATHIONE REDUCTASE1 (GR1) or NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), and were linked to compromised induction of ISOCHORISMATE SYNTHASE1 (ICS1) and ICS1-dependent SA accumulation. INNOVATION: A novel genetic approach was used in which GSH content or antioxidative capacity was independently modified in an H(2)O(2) signaling background. Analysis of new double and triple mutants allowed us to infer previously undescribed regulatory roles for GSH. CONCLUSION: In parallel to its antioxidant role, GSH acts independently of NPR1 to allow increased intracellular H(2)O(2) to activate SA signaling, a key defense response in plants.

Regulation of basal and oxidative stress-triggered jasmonic acid-related gene expression by glutathione.

Glutathione is a determinant of cellular redox state with roles in defence and detoxification. Emerging concepts suggest that this compound also has functions in cellular signalling. Here, we report evidence that glutathione plays potentially important roles in setting signalling strength through the jasmonic acid (JA) pathway. Firstly, we show that basal expression of JA-related genes is correlated with leaf glutathione content when the latter is manipulated either genetically or pharmacologically. Secondly, analyses of an oxidative stress signalling mutant, cat2, reveal that up-regulation of the JA pathway triggered by intracellular oxidation requires accompanying glutathione accumulation. Genetically blocking this accumulation in a cat2 cad2 line largely annuls H2 O2 -induced expression of JA-linked genes, and this effect can be rescued by exogenously supplying glutathione. While most attention on glutathione functions in biotic stress responses has been focused on the thiol-regulated protein NPR1, a comparison of JA-linked gene expression in cat2 cad2 and cat2 npr1 double mutants provides evidence that glutathione acts through other components to regulate the response of this pathway to oxidative stress. Our study provides new information implicating glutathione as a factor determining basal JA gene expression and suggests novel glutathione-dependent control points that regulate JA signalling in response to intracellular oxidation.

AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis.

This work investigated the contribution of AtRbohD and AtRbohF to regulating defence-associated metabolism during three types of interaction: (i) incompatible and (ii) compatible interaction with Pseudomonas syringae; and (iii) intracellular oxidative stress in the catalase-deficient cat2 background. In all three cases, loss of function of either gene modulated the response of defence compounds. AtRbohF gene function was necessary for rapid and full induction of salicylic acid (SA) during compatible and incompatible interactions, and for resistance to virulent bacteria. Both artrboh mutations modulated the effects of intracellular ROS in the cat2 background, although the predominant effect was mediated by atrbohF. Loss of this gene function increased lesion formation in cat2 but uncoupled this effect from cat2-triggered induction of SA and camalexin, accumulation of glutathione and disease resistance, all of which were much lower in cat2 artbohF than in cat2. A detailed comparison of GC-TOF-MS profiles produced by the three interactions revealed considerable overlap between cat2 effects and those produced by bacterial infection in the wild-type background. Analysis of the impact of the two atrboh mutations on these profiles provided further evidence that AtRbohF interacts closely with intracellular oxidative stress to tune dynamic metabolic responses during infection. Thus, AtRbohF appears to be a key player not only in HR-related cell death but also in regulating metabolomic responses and resistance. Based on the results obtained during the three types of interaction, a model is proposed of how NADPH oxidases and intracellular ROS interact to determine the outcome of pathogen defence responses.

Glutathione in plants: an integrated overview.

Plants cannot survive without glutathione (γ-glutamylcysteinylglycine) or γ-glutamylcysteine-containing homologues. The reasons why this small molecule is indispensable are not fully understood, but it can be inferred that glutathione has functions in plant development that cannot be performed by other thiols or antioxidants. The known functions of glutathione include roles in biosynthetic pathways, detoxification, antioxidant biochemistry and redox homeostasis. Glutathione can interact in multiple ways with proteins through thiol-disulphide exchange and related processes. Its strategic position between oxidants such as reactive oxygen species and cellular reductants makes the glutathione system perfectly configured for signalling functions. Recent years have witnessed considerable progress in understanding glutathione synthesis, degradation and transport, particularly in relation to cellular redox homeostasis and related signalling under optimal and stress conditions. Here we outline the key recent advances and discuss how alterations in glutathione status, such as those observed during stress, may participate in signal transduction cascades. The discussion highlights some of the issues surrounding the regulation of glutathione contents, the control of glutathione redox potential, and how the functions of glutathione and other thiols are integrated to fine-tune photorespiratory and respiratory metabolism and to modulate phytohormone signalling pathways through appropriate modification of sensitive protein cysteine residues.

Increased intracellular H2O2 availability preferentially drives glutathione accumulation in vacuoles and chloroplasts.

One biochemical response to increased H2O2 availability is the accumulation of glutathione disulphide (GSSG), the disulphide form of the key redox buffer glutathione. It remains unclear how this potentially important oxidative stress response impacts on the different sub-cellular glutathione pools. We addressed this question by using two independent in situ glutathione labelling techniques in Arabidopsis wild type (Col-0) and the GSSG-accumulating cat2 mutant. A comparison of in situ labelling with monochlorobimane (MCB) and in vitro labelling with monobromobimane (MBB) revealed that, whereas in situ labelling of Col-0 leaf glutathione was complete within 2 h incubation, about 50% of leaf glutathione remained inaccessible to MCB in cat2. High-performance liquid chromatography (HPLC) and enzymatic assays showed that this correlated tightly with the glutathione redox state, pointing to significant in vivo pools of GSSG in cat2 that were unavailable for MCB labelling. Immunogold labelling of leaf sections to estimate sub-cellular glutathione distribution showed that the accumulated GSSG in cat2 was associated with only a minor increase in cytosolic glutathione but with a 3- and 10-fold increase in plastid and vacuolar pools, respectively. The data are used to estimate compartment-specific glutathione concentrations under optimal and oxidative stress conditions, and the implications for redox homeostasis and signalling are discussed.