Reactive oxygen species (ROS) are central regulators of plant growth and stress responses. Cellular ROS levels are tightly controlled by antioxidant systems, including the evolutionarily conserved catalases that detoxify hydrogen peroxide (H 2 O 2 ) predominantly within peroxisomes. Despite their importance, substantial gaps remain in our understanding of catalase biogenesis, regulation, subcellular targeting, and potential extra-peroxisomal functions. Using affinity purification of the UV-B photoreceptor UVR8 coupled with mass spectrometry, we identified a REGULATOR OF CHROMATIN CONDENSATION 1–like protein in Arabidopsis , which we named CATALASE-INTERACTING RCC1-LIKE 1 (CAIR1). CAIR1 interacts with all three catalase isoforms (CAT1–CAT3) as well as their chaperone NO CATALASE ACTIVITY 1 (NCA1). Loss-of-function cair1 mutants partially phenocopy cat2 and nca1 , with reduced catalase activity, enhanced sensitivity to oxidative stress and alkaline growth conditions, and impaired primary root elongation. Mechanistically, cytosolic interaction between CAIR1 and CAT2 enhances total cellular catalase activity by facilitating peroxisomal import and proper subcellular localization of CAT2. In the absence of CAIR1, CAT2 forms aggregates, likely accounting for the observed loss of catalase activity. Notably, CAIR1 undergoes reversible, redox-dependent oligomerization that enhances its interaction with catalases. Mutation of CAIR1 at Cys-356 and Cys-545 compromises this interaction under elevated ROS conditions and fails to rescue the oxidative stress sensitivity of cair1 mutants. Moreover, UV-B exposure suppresses catalase activity by weakening the interaction between CAIR1 and catalases, thus linking environmental light signalling to cellular redox regulation. Together, our findings reveal CAIR1 as a dynamic redox-responsive regulator of catalase activity that maintains cellular redox homeostasis by coordinating catalase localization and function through reversible oligomerization.

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.

Grass pea (Lathyrus sativus L.) is a grain legume of increasing importance in the Mediterranean region due to its outstanding tolerance to abiotic stresses such as salinity, heat, drought, and flooding, outperforming many other legume species. Despite established natural phenotypic variation in response to water-related stresses, the genetic basis of this resilience remains poorly understood, hindering precision breeding for single and combined stress tolerance. A genome-wide association study was conducted here to investigate the genetic architecture of water stress responses in grass pea. Previously, phenotypic data, including gas exchange, chlorophyll a fluorescence, photosynthetic pigments, leaf water status, and biomass partitioning traits, were assessed under well-watered, mild drought, and partial submergence conditions across 194 representative grass pea accessions worldwide. The data were associated with 5,651 single nucleotide polymorphisms (SNPs) using linear mixed models under a restricted maximum likelihood framework, incorporating population structure and the newly assembled L0007 genome. A total of 130 unique SNPs associated with at least one trait-treatment combination or with trait variation between stress and control conditions, providing a valuable resource for precision breeding of multi-stress tolerance in grass pea. The loci associated with drought and waterlogging were largely non-overlapping, suggesting distinct genetic bases for the two stress tolerances. However, some common mechanisms, such as redox regulation and carbohydrate metabolism, emerged among the identified candidate genes, highlighting some interconnectedness of biological pathways involved in grass pea responses to water stress.

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.

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.

In aerobic life forms, reactive oxygen species (ROS) are produced by the partial reduction of oxygen during energy-generating metabolic processes. In plants, ROS production increases during periods of both abiotic and biotic stress, severely overloading the antioxidant systems. Hydrogen peroxide (H2O2) plays a central role in cellular redox homeostasis and signalling by oxidizing crucial cysteines to sulfenic acid, which is considered a biologically relevant post-translational modification (PTM). Until now, the impact of the nucleus on cellular redox homeostasis has been relatively unexplored. The regulation of histone-modifying enzymes by oxidative PTMs at redox-sensitive cysteine or tyrosine residues is particularly intriguing because it allows the integration of redox signalling mechanisms with chromatin control of transcriptional activity. One of the most extensively studied histone acetyltransferases is the conserved GENERAL CONTROL NONDEPRESSIBLE 5 (GCN5) complex. This study investigated the nuclear sulfenome in Arabidopsis thaliana by expressing a nuclear variant of the Yeast Activation Protein-1 (YAP1) probe and identified 225 potential redox-active proteins undergoing S-sulfenylation. Mass spectrometry analysis further confirmed the S-sulfenylation of GCN5 at Cys293, Cys368, and Cys400, and their functional significance and impact on the GCN5 protein-protein interaction network were assessed using cysteine-to-serine mutagenesis.

Redox signalling is crucial for regulating plant development and adaptation to environmental changes. Proteins with redox-sensitive cysteines can sense oxidative stress and modulate their functions. Recent proteomics efforts have comprehensively mapped the proteins targeted by oxidative modifications. The nucleus, the epicentre of transcriptional reprogramming, contains a large number of proteins that control gene expression. Specific redox-sensitive transcription factors have long been recognized as key players in decoding redox signals in the nucleus and thus in regulating transcriptional responses. Consequently, the redox regulation of the nuclear transcription machinery and its cofactors has received less attention. In this review, we screened proteomic datasets for redox-sensitive cysteines on proteins of the core transcription complexes and chromatin modifiers in Arabidopsis thaliana. Our analysis indicates that redox regulation affects every step of gene transcription, from initiation to elongation and termination. We report previously undescribed redox-sensitive subunits in transcription complexes and discuss the emerging challenges in unravelling the landscape of redox-regulated processes involved in nuclear gene transcription. This review highlights essential transcription machinery complexes and chromatin modifiers targeted by redox regulation, filling knowledge gaps in the intricate field of gene transcription.

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.

Flooding impairs plant growth through oxygen deprivation, which activates plant survival and acclimation responses. Transcriptional responses to low oxygen are generally associated with the activation of group VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors. However, the exact mechanisms and molecular components by which ERFVII factors initiate gene expression are not fully elucidated. Here, we show that the ERFVII factors RELATED TO APETALA 2.2 (RAP2.2) and RAP2.12 cooperate with the Mediator complex subunit AtMED25 to coordinate gene expression under hypoxia in Arabidopsis thaliana. Respective med25 knock-out mutants display reduced low-oxygen stress tolerance. AtMED25 physically associates with a distinct set of hypoxia core genes and its loss partially impairs transcription under hypoxia due to decreased RNA polymerase II recruitment. Association of AtMED25 with target genes requires the presence of ERFVII transcription factors. Next to ERFVII protein stabilisation, also the composition of the Mediator complex including AtMED25 is potentially affected by hypoxia stress as shown by protein-complex pulldown assays. The dynamic response of the Mediator complex to hypoxia is furthermore supported by the fact that two subunits, AtMED8 and AtMED16, are not involved in the establishment of hypoxia tolerance, whilst both act in coordination with AtMED25 under other environmental conditions. We furthermore show that AtMED25 function under hypoxia is independent of ethylene signalling. Finally, functional conservation at the molecular level was found for the MED25-ERFVII module between A. thaliana and the monocot species Oryza sativa, pointing to a potentially universal role of MED25 in coordinating ERFVII-dependent transcript responses to hypoxia in plants.

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.

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.

Hydrogen peroxide (H2O2) is a molecule generated by plant cells during vital processes such as photosynthesis and photorespiration. This molecule is required for plant development and growth, as well as for coordinating appropriate responses to survive in a changing climate. Much attention has been dedicated to studying the functions of H2O2 in plant abiotic and biotic stress responses, mainly because of its role as a signal and its close interaction with phytohormones and redox-dependent signaling pathways. Like other reactive oxygen species (ROS), H2O2 has a higher energy state than oxygen which facilitate its interactions with cellular components, these include metabolites that are required to control the levels of H2O2 such as ascorbate and glutathione. The functions of H2O2 depend on its intracellular concentrations and these are regulated, among other factors, by enzymes that metabolize H2O2 to water and oxygen such as catalases and ascorbate peroxidases. H2O2 triggers signaling via oxidizing Methionine and Cysteine residues on different proteins, some acting as sensors that help to amplify and transduce H2O2 signals. In this Chapter, I provide a comprehensive overview of cellular components that are involved in H2O2 generation and processing. I also discuss the different ways by which H2O2 can act as a signal and summarize recent discoveries of H2O2 sensors and receptors that link H2O2 to other signal transduction pathways.

Plant cells sense fluctuations in the levels of cellular polyamines (PAs), namely, putrescine (Put), spermidine (Spd) and spermine (Spm).PA cellular titer regulates biological processes, including senescence.Changes in PA concentration in properly functioning cells are unlikely.Induced senescence disturbs their homeostasis which leads to senescence-dependent metabolic changes.PA catabolism was proposed as process promoting mainly by H2O2 production.Here, we show that darkness-induced plant senescence is associated with changes in the PA levels together with the changes of signaling molecules such as nitric oxide (NO) and hydrogen peroxide (H2O2).Upon blocking Put catabolism, the senescing cells generated lesser amounts of NO and more of H2O2 and, in turn, accelerated senescence.We opine that a possible linkage exists between PAs and signaling molecules such as NO and H2O2.Thus, the balance in the triad network made of NO↔PA↔H2O2 signaling may be involved in re-programming metabolism and regulate the direction in which a plant may be ushered into -growth, senescence or cell death.

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.

regulatory interactions, were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions) in this work.The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs.For regulators predicted to be involved in reactive oxygen species stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses.This includes 13 novel ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss-or gain-of-function lines (MV, H2O2 or 3-AT).In conclusion, the presented iGRN offers a high-quality starting point to give us a better understanding for gene regulation in plants by integrating different experimental data types at the network level.

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.

Signaling events triggered by hydrogen peroxide (H2O2) regulate plant growth and defense by orchestrating a genome-wide transcriptional reprogramming. However, the specific mechanisms that govern H2O2-dependent gene expression are still poorly understood. Here, we identify the Arabidopsis Mediator complex subunit MED8 as a regulator of H2O2 responses. The introduction of the med8 mutation in a constitutive oxidative stress genetic background (catalase-deficient, cat2) was associated with enhanced activation of the salicylic acid pathway and accelerated cell death. Interestingly, med8 seedlings were more tolerant to oxidative stress generated by the herbicide methyl viologen (MV) and exhibited transcriptional hyperactivation of defense signaling, in particular salicylic acid- and jasmonic acid-related pathways. The med8-triggered tolerance to MV was manipulated by the introduction of secondary mutations in salicylic acid and jasmonic acid pathways. In addition, analysis of the Mediator interactome revealed interactions with components involved in mRNA processing and microRNA biogenesis, hence expanding the role of Mediator beyond transcription. Notably, MED8 interacted with the transcriptional regulator NEGATIVE ON TATA-LESS, NOT2, to control the expression of H2O2-inducible genes and stress responses. Our work establishes MED8 as a component regulating oxidative stress responses and demonstrates that it acts as a negative regulator of H2O2-driven activation of defense gene expression.