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.

Redox regulation of gene expression: proteomics reveals multiple previously undescribed redox-sensitive cysteines in transcription complexes and chromatin modifiers.

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 recognised 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.

Roadmap for the next decade of plant programmed cell death research.

Programmed cell death (PCD) is fundamentally important for plant development, abiotic stress responses and immunity, but our understanding of its regulation remains fragmented. Building a stronger research community is required to accelerate progress in this area through knowledge exchange and constructive debate. In this Viewpoint, we aim to initiate a collective effort to integrate data across a diverse set of experimental models to facilitate characterisation of the fundamental mechanisms underlying plant PCD and ultimately aid the development of a new plant cell death classification system in the future. We also put forward our vision for the next decade of plant PCD research stemming from discussions held during the 31st New Phytologist workshop, 'The Life and Death Decisions of Plant Cells' that took place at University College Dublin in Ireland (14-15 June 2023). We convey the key areas of significant progress and possible future research directions identified, including resolving the spatiotemporal control of cell death, isolation of its molecular and genetic regulators, and harnessing technical advances for studying PCD events in plants. Further, we review the breadth of potential impacts of plant PCD research and highlight the promising new applications of findings from this dynamically evolving field.

Redox regulation of chromatin remodelling in plants.

Changes in the cellular redox balance that occur during plant responses to unfavourable environmental conditions significantly affect a myriad of redox-sensitive processes, including those that impact on the epigenetic state of the chromatin. Various epigenetic factors, like histone modifying enzymes, chromatin remodelers, and DNA methyltransferases can be targeted by oxidative posttranslational modifications. As their combined action affects the epigenetic regulation of gene expression, they form an integral part of plant responses to (a)biotic stress. Epigenetic changes triggered by unfavourable environmental conditions are intrinsically linked with primary metabolism that supplies intermediates and donors, such acetyl-CoA and S-adenosyl-methionine, that are critical for the epigenetic decoration of histones and DNA. Here, we review the recent advances in our understanding of redox regulation of chromatin remodelling, dynamics of epigenetic marks, and the interplay between epigenetic control of gene expression, redox signalling and primary metabolism within an (a)biotic stress context.

Cysteine thiol sulfinic acid in plant stress signaling.

Cysteine thiols are susceptible to various oxidative posttranslational modifications (PTMs) due to their high chemical reactivity. Thiol-based PTMs play a crucial role in regulating protein functions and are key contributors to cellular redox signaling. Although reversible thiol-based PTMs, such as disulfide bond formation, S-nitrosylation, and S-glutathionylation, have been extensively studied for their roles in redox regulation, thiol sulfinic acid (-SO2 H) modification is often perceived as irreversible and of marginal significance in redox signaling. Here, we revisit this narrow perspective and shed light on the redox regulatory roles of -SO2 H in plant stress signaling. We provide an overview of protein sulfinylation in plants, delving into the roles of hydrogen peroxide-mediated and plant cysteine oxidase-catalyzed formation of -SO2 H, highlighting the involvement of -SO2 H in specific regulatory signaling pathways. Additionally, we compile the existing knowledge of the -SO2 H reducing enzyme, sulfiredoxin, offering insights into its molecular mechanisms and biological relevance. We further summarize current proteomic techniques for detecting -SO2 H and furnish a list of experimentally validated cysteine -SO2 H sites across various species, discussing their functional consequences. This review aims to spark new insights and discussions that lead to further investigations into the functional significance of protein -SO2 H-based redox signaling in plants.

CysQuant: Simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry.

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

H2O2-dependent oxidation of the transcription factor GmNTL1 promotes salt tolerance in soybean.

Reactive oxygen species (ROS) play an essential role in plant growth and responses to environmental stresses. Plant cells sense and transduce ROS signaling directly via hydrogen peroxide (H2O2)-mediated posttranslational modifications (PTMs) on protein cysteine residues. Here, we show that the H2O2-mediated cysteine oxidation of NAC WITH TRANS-MEMBRANE MOTIF1-LIKE 1 (GmNTL1) in soybean (Glycine max) during salt stress promotes its release from the endoplasmic reticulum (ER) membrane and translocation to the nucleus. We further show that an oxidative posttranslational modification on GmNTL1 residue Cys-247 steers downstream amplification of ROS production by binding to and activating the promoters of RESPIRATORY BURST OXIDASE HOMOLOG B (GmRbohB) genes, thereby creating a feed-forward loop to fine-tune GmNTL1 activity. In addition, oxidation of GmNTL1 Cys-247 directly promotes the expression of CATION H+ EXCHANGER 1 (GmCHX1)/SALT TOLERANCE-ASSOCIATED GENE ON CHROMOSOME 3 (GmSALT3) and Na+/H+ Antiporter 1 (GmNHX1). Accordingly, transgenic overexpression of GmNTL1 in soybean increases the H2O2 levels and K+/Na+ ratio in the cell, promotes salt tolerance, and increases yield under salt stress, while an RNA interference-mediated knockdown of GmNTL1 elicits the opposite effects. Our results reveal that the salt-induced oxidation of GmNTL1 promotes its relocation and transcriptional activity through an H2O2-mediated posttranslational modification on cysteine that improves resilience of soybean against salt stress.

A phloem-localized Arabidopsis metacaspase (AtMC3) improves drought tolerance.

Increasing drought phenomena pose a serious threat to agricultural productivity. Although plants have multiple ways to respond to the complexity of drought stress, the underlying mechanisms of stress sensing and signaling remain unclear. The role of the vasculature, in particular the phloem, in facilitating inter-organ communication is critical and poorly understood. Combining genetic, proteomic and physiological approaches, we investigated the role of AtMC3, a phloem-specific member of the metacaspase family, in osmotic stress responses in Arabidopsis thaliana. Analyses of the proteome in plants with altered AtMC3 levels revealed differential abundance of proteins related to osmotic stress pointing into a role of the protein in water-stress-related responses. Overexpression of AtMC3 conferred drought tolerance by enhancing the differentiation of specific vascular tissues and maintaining higher levels of vascular-mediated transportation, while plants lacking the protein showed an impaired response to drought and inability to respond effectively to the hormone abscisic acid. Overall, our data highlight the importance of AtMC3 and vascular plasticity in fine-tuning early drought responses at the whole plant level without affecting growth or yield.

Structure-function study of a Ca2+-independent metacaspase involved in lateral root emergence.

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. As the structure-function relationship of metacaspases remains poorly understood, we solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) belonging to a particular subgroup not requiring calcium ions for activation. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors and found several hits with a minimal thioxodihydropyrimidine-dione structure, of which some are specific AtMCA-IIf inhibitors. We provide mechanistic insight into the basis of inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) effectively hampered lateral root emergence in vivo, probably through inhibition of metacaspases specifically expressed in the endodermal cells overlying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in other species, such as important human pathogens, including those causing neglected diseases.

Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience.

Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Smith (Sm) antigen protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of the mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, as for sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and in enhanced resilience to oxidative 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.

Mechanisms controlling plant proteases and their substrates.

In plants, proteolysis is emerging as an important field of study due to a growing understanding of the critical involvement of proteases in plant cell death, disease and development. Because proteases irreversibly modify the structure and function of their target substrates, proteolytic activities are stringently regulated at multiple levels. Most proteases are produced as dormant isoforms and only activated in specific conditions such as altered ion fluxes or by post-translational modifications. Some of the regulatory mechanisms initiating and modulating proteolytic activities are restricted in time and space, thereby ensuring precision activity, and minimizing unwanted side effects. Currently, the activation mechanisms and the substrates of only a few plant proteases have been studied in detail. Most studies focus on the role of proteases in pathogen perception and subsequent modulation of the plant reactions, including the hypersensitive response (HR). Proteases are also required for the maturation of coexpressed peptide hormones that lead essential processes within the immune response and development. Here, we review the known mechanisms for the activation of plant proteases, including post-translational modifications, together with the effects of proteinaceous inhibitors.

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.

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures.

Deep learning algorithms such as AlphaFold2 predict three-dimensional protein structure with high confidence. The recent release of more than 200 million structural models provides an unprecedented resource for functional protein annotation. Here, we used AlphaFold2 predicted structures of fifteen plant proteomes to functionally and evolutionary analyze cysteine residues in the plant kingdom. In addition to identification of metal ligands coordinated by cysteine residues, we systematically analyzed cysteine disulfides present in these structural predictions. Our analysis demonstrates most of these predicted disulfides are trustworthy due their high agreement (∼96%) with those present in X-ray and NMR protein structures, their characteristic disulfide stereochemistry, the biased subcellular distribution of their proteins and a higher degree of oxidation of their respective cysteines as measured by proteomics. Adopting an evolutionary perspective, zinc binding sites are increasingly present at the expense of iron-sulfur clusters in plants. Interestingly, disulfide formation is increased in secreted proteins of land plants, likely promoting sequence evolution to adapt to changing environments encountered by plants. In summary, Alphafold2 predicted structural models are a rich source of information for studying the role of cysteines residues in proteins of interest and for protein redox biology in general.

Detection and Quantification of Protein Aggregates in Plants.

Protein quality control is an important aspect of stress recovery. It maintains protein homeostasis through a machinery of regulatory proteins such as chaperones and proteases. When the system recognizes accumulation of misfolded or aggregated proteins, the cell recruits a set of regulatory proteins to initiate protein quality control. To understand the dynamics of stress-mediated aggregate protein formation and recovery in plants, robust methods aimed at detecting and measuring such protein aggregates are needed. This will help us to deepen our understanding of protein quality control mechanisms in plants.

Cysteine thiol-based post-translational modification: What do we know about transcription factors?

Reactive electrophilic species are ubiquitous in plant cells, where they contribute to specific redox-regulated signaling events. Redox signaling is known to modulate gene expression during diverse biological processes, including plant growth, development, and environmental stress responses. Emerging data demonstrates that transcription factors (TFs) are a main target of cysteine thiol-based oxidative post-translational modifications (OxiPTMs), which can alter their transcriptional activity and thereby convey redox information to the nucleus. Here, we review the significant progress that has been made in characterizing cysteine thiol-based OxiPTMs, their biochemical properties, and their functional effects on plant TFs. We discuss the underlying mechanism of redox regulation and its contribution to various physiological processes as well as still outstanding challenges in redox regulation of plant gene expression.

To Be or Not to Be? Are Reactive Oxygen Species, Antioxidants, and Stress Signalling Universal Determinants of Life or Death?

In the environmental and organism context, oxidative stress is complex and unavoidable. Organisms simultaneously cope with a various combination of stress factors in natural conditions. For example, excess light stress is accompanied by UV stress, heat shock stress, and/or water stress. Reactive oxygen species (ROS) and antioxidant molecules, coordinated by electrical signalling (ES), are an integral part of the stress signalling network in cells and organisms. They together regulate gene expression to redirect energy to growth, acclimation, or defence, and thereby, determine cellular stress memory and stress crosstalk. In plants, both abiotic and biotic stress increase energy quenching, photorespiration, stomatal closure, and leaf temperature, while toning down photosynthesis and transpiration. Locally applied stress induces ES, ROS, retrograde signalling, cell death, and cellular light memory, then acclimation and defence responses in the local organs, whole plant, or even plant community (systemic acquired acclimation, systemic acquired resistance, network acquired acclimation). A simplified analogy can be found in animals where diseases vs. fitness and prolonged lifespan vs. faster aging, are dependent on mitochondrial ROS production and ES, and body temperature is regulated by sweating, temperature-dependent respiration, and gene regulation. In this review, we discuss the universal features of stress factors, ES, the cellular production of ROS molecules, ROS scavengers, hormones, and other regulators that coordinate life and death.

ROS and redox regulation of cell-to-cell and systemic signaling in plants during stress.

Stress results in the enhanced accumulation of reactive oxygen species (ROS) in plants, altering the redox state of cells and triggering the activation of multiple defense and acclimation mechanisms. In addition to activating ROS and redox responses in tissues that are directly subjected to stress (termed 'local' tissues), the sensing of stress in plants triggers different systemic signals that travel to other parts of the plant (termed 'systemic' tissues) and activate acclimation and defense mechanisms in them; even before they are subjected to stress. Among the different systemic signals triggered by stress in plants are electric, calcium, ROS, and redox waves that are mobilized in a cell-to-cell fashion from local to systemic tissues over long distances, sometimes at speeds of up to several millimeters per second. Here, we discuss new studies that identified various molecular mechanisms and proteins involved in mediating systemic signals in plants. In addition, we highlight recent studies that are beginning to unravel the mode of integration and hierarchy of the different systemic signals and underline open questions that require further attention. Unraveling the role of ROS and redox in plant stress responses is highly important for the development of climate resilient crops.