Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling

Glutathione has numerous roles in cellular defence and in sulphur metabolism. These functions depend or impact on the concentration and/or redox state of leaf glutathione pools. Effective function requires homeostatic control of concentration and redox state, with departures from homeostasis acting as signals that trigger adaptive responses. Intercellular and intracellular glutathione pools are linked by transport across membranes. It is shown that glutathione can cross the chloroplast envelope at rates similar to the speed of biosynthesis. Control of glutathione concentration and redox state is therefore due to a complex interplay between biosynthesis, utilization, degradation, oxidation/reduction, and transport. All these factors must be considered in order to evaluate the significance of glutathione as a signalling component during development, abiotic stress, or pathogen attack.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E(GSSG/2 GSH)) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E(CySS/2 Cys)). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a 'thiol-disulphide redox environment' (E(thiol-disulphide)), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E(CySS/2 Cys) to E(thiol-disulphide) in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Glutathione,photosynthesis and the redox regulation of stress-responsive gene expression

The ubiquitous antioxidant thiol tripeptide glutathione is present in millimolar concentrations in plant tissues and is regarded as one of the major determinants of cellular redox homeostasis. Recent research has highlighted a regulatory role for glutathione in influencing the expression of many genes important in plants' responses to both abiotic and biotic stress. Therefore, it becomes important to consider how glutathione levels and its redox state are influenced by environmental factors, how glutathione is integrated into primary metabolism and precisely how it can influence the functioning of signal transduction pathways by modulating cellular redox state. This review draws on a number of recent important observations and papers to present a unified view of how the responsiveness of glutathione to changes in photosynthesis may be one means of linking changes in nuclear gene expression to changes in the plant's external environment.     

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 H₂O₂‐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.     

Glutathione and modulation of cell apoptosis

Apoptosis is a highly organized form of cell death that is important for tissue homeostasis, organ development and senescence. To date, the extrinsic (death receptor mediated) and intrinsic (mitochondria derived) apoptotic pathways have been characterized in mammalian cells. Reduced glutathione, is the most prevalent cellular thiol that plays an essential role in preserving a reduced intracellular environment. glutathione protection of cellular macromolecules like deoxyribose nucleic acid proteins and lipids against oxidizing, environmental and cytotoxic agents, underscores its central anti-apoptotic function. Reactive oxygen and nitrogen species can oxidize cellular glutathione or induce its extracellular export leading to the loss of intracellular redox homeostasis and activation of the apoptotic signaling cascade. Recent evidence uncovered a novel role for glutathione involvement in apoptotic signaling pathways wherein post-translational S-glutathiolation of protein redox active cysteines is implicated in the potentiation of apoptosis. In the present review we focus on the key aspects of glutathione redox mechanisms associated with apoptotic signaling that includes: (a) changes in cellular glutathione redox homeostasis through glutathione oxidation or GSH transport in relation to the initiation or propagation of the apoptotic cascade, and (b) evidence for S-glutathiolation in protein modulation and apoptotic initiation.     

The functions of inter- and intracellular glutathione transport systems in plants

Glutathione is one of the major redox buffers in most aerobic cells, and it has a broad spectrum of functions in plants. Recent discoveries implicate this thiol peptide in signalling and cellular homeostasis. Glutathione can sense intracellular redox status: perturbations of glutathione reduction state are transduced into changes in gene expression. This central role demands precise control of both the concentration and the reduction state of glutathione in different compartments. In addition to the regulation of glutathione biosynthesis and redox state, attention is now turning to the role of glutathione transporters.     

Molecular Mechanisms of Glutaredoxin Enzymes: Versatile Hubs for Thiol–Disulfide Exchange between Protein Thiols and Glutathione

The tripeptide glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) constitute a key redox couple in cells. In particular, they partner protein thiols in reversible thiol–disulfide exchange reactions that act as switches in cell signaling and redox homeostasis. Disruption of these processes may impair cellular redox signal transduction and induce redox misbalances that are linked directly to aging processes and to a range of pathological conditions including cancer, cardiovascular diseases and neurological disorders. Glutaredoxins are a class of GSH-dependent oxidoreductase enzymes that specifically catalyze reversible thiol–disulfide exchange reactions between protein thiols and the abundant thiol pool GSSG/GSH. They protect protein thiols from irreversible oxidation, regulate their activities under a variety of cellular conditions and are key players in cell signaling and redox homeostasis. On the other hand, they may also function as metal-binding proteins with a possible role in the cellular homeostasis and metabolism of essential metals copper and iron. However, the molecular basis and underlying mechanisms of glutaredoxin action remain elusive in many situations. This review focuses specifically on these aspects in the context of recent developments that illuminate some of these uncertainties.     

Redox state of low-molecular-weight thiols and disulphides during somatic embryogenesis of salt-treated suspension cultures of Dactylis glomerata L.

Abstract

The tripeptide antioxidant γ-L-glutamyl-L-cysteinyl-glycine, or glutathione (GSH), serves a central role in ROS scavenging and oxidative signalling. Here, GSH, glutathione disulphide (GSSG), and other low-molecular-weight (LMW) thiols and their corresponding disulphides were studied in embryogenic suspension cultures of Dactylis glomerata L. subjected to moderate (0.085 M NaCl) or severe (0.17 M NaCl) salt stress. Total glutathione (GSH + GSSG) concentrations and redox state were associated with growth and development in control cultures and in moderately salt-stressed cultures and were affected by severe salt stress. The redox state of the cystine (CySS)/2 cysteine (Cys) redox couple was also affected by developmental stage and salt stress. The glutathione half-cell reduction potential (E_{GSSG/2 GSH}) increased with the duration of culturing and peaked when somatic embryos were formed, as did the half-cell reduction potential of the CySS/2 Cys redox couple (E_{CySS/2 Cys}). The most noticeable relationship between cellular redox state and developmental state was found when all LMW thiols and disulphides present were mathematically combined into a ‘thiol–disulphide redox environment’ (E_{thiol–disulphide}), whereby reducing conditions accompanied proliferation, resulting in the formation of pro-embryogenic masses (PEMs), and oxidizing conditions accompanied differentiation, resulting in the formation of somatic embryos. The comparatively high contribution of E_{CySS/2 Cys} to E_{thiol–disulphide} in cultures exposed to severe salt stress suggests that Cys and CySS may be important intracellular redox regulators with a potential role in stress signalling.     

Free radicals and antioxidants in normal physiological functions and human disease

Reactive oxygen species (ROS) and reactive nitrogen species (RNS, e.g. nitric oxide, NO(*)) are well recognised for playing a dual role as both deleterious and beneficial species. ROS and RNS are normally generated by tightly regulated enzymes, such as NO synthase (NOS) and NAD(P)H oxidase isoforms, respectively. Overproduction of ROS (arising either from mitochondrial electron-transport chain or excessive stimulation of NAD(P)H) results in oxidative stress, a deleterious process that can be an important mediator of damage to cell structures, including lipids and membranes, proteins, and DNA. In contrast, beneficial effects of ROS/RNS (e.g. superoxide radical and nitric oxide) occur at low/moderate concentrations and involve physiological roles in cellular responses to noxia, as for example in defence against infectious agents, in the function of a number of cellular signalling pathways, and the induction of a mitogenic response. Ironically, various ROS-mediated actions in fact protect cells against ROS-induced oxidative stress and re-establish or maintain "redox balance" termed also "redox homeostasis". The "two-faced" character of ROS is clearly substantiated. For example, a growing body of evidence shows that ROS within cells act as secondary messengers in intracellular signalling cascades which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. This review will describe the: (i) chemistry and biochemistry of ROS/RNS and sources of free radical generation; (ii) damage to DNA, to proteins, and to lipids by free radicals; (iii) role of antioxidants (e.g. glutathione) in the maintenance of cellular "redox homeostasis"; (iv) overview of ROS-induced signaling pathways; (v) role of ROS in redox regulation of normal physiological functions, as well as (vi) role of ROS in pathophysiological implications of altered redox regulation (human diseases and ageing). Attention is focussed on the ROS/RNS-linked pathogenesis of cancer, cardiovascular disease, atherosclerosis, hypertension, ischemia/reperfusion injury, diabetes mellitus, neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), rheumatoid arthritis, and ageing. Topics of current debate are also reviewed such as the question whether excessive formation of free radicals is a primary cause or a downstream consequence of tissue injury.     

ROS and redox signalling in the response of plants to abiotic stress

The redox state of the chloroplast and mitochondria, the two main powerhouses of photosynthesizing eukaryotes, is maintained by a delicate balance between energy production and consumption, and affected by the need to avoid increased production of reactive oxygen species (ROS). These demands are especially critical during exposure to extreme environmental conditions, such as high light (HL) intensity, heat, drought or a combination of different environmental stresses. Under these conditions, ROS and redox cues, generated in the chloroplast and mitochondria, are essential for maintaining normal energy and metabolic fluxes, optimizing different cell functions, activating acclimation responses through retrograde signalling, and controlling whole-plant systemic signalling pathways. Regulation of the multiple redox and ROS signals in plants requires a high degree of coordination and balance between signalling and metabolic pathways in different cellular compartments. In this review, we provide an update on ROS and redox signalling in the context of abiotic stress responses, while addressing their role in retrograde regulation, systemic acquired acclimation and cellular coordination in plants.     

Interactions between hormone and redox signalling pathways in the control of growth and cross tolerance to stress

The ability of plants to respond to a wide range of environmental stresses is highly flexible and finely balanced through the interaction of hormonal plant growth regulators and the redox signalling hub, which integrates information from the environment and cellular metabolism/physiology. Plant hormones produce reactive oxygen species (ROS) as second messengers in signalling cascades that convey information concerning changes in hormone concentrations and/or sensitivity to mediate a whole range of adaptive responses. Cellular redox buffering capacity that is determined largely by the abundance of ascorbate has a profound influence on the threshold at which hormone signalling is triggered and on the interactions between different hormones. Other antioxidants such as glutathione, glutaredoxins and thioredoxins are also central redox regulators of hormone signalling pathways. The complex network of cross-communication between oxidants and antioxidants in the redox signalling hub and the different hormone signalling pathways maximises productivity under stress-free situations and regulates plant growth, development, reproduction, programmed cell death and survival upon exposure to stress. This interactive network confers enormous regulatory potential because it allows plants to adapt to changing and often challenging conditions, while preventing boom or bust scenarios with regard to resources, ensuring that energy is produced and utilised in a safe and efficient manner.     

Oxidative Protein Folding in the Secretory Pathway and Redox Signaling Across Compartments and Cells

The endoplasmic reticulum (ER) is central for many essential cellular activities, such as folding, assembly and quality control of secretory and membrane proteins, disulfide bond formation, glycosylation, lipid biosynthesis, Ca²⁺ storage and signaling. In addition, this multifunctional organelle integrates many adaptive and/or maladaptive signaling cues reporting on metabolism, proteostasis, Ca²⁺ and redox homeostasis. We are beginning to understand how these functions and pathways are integrated with one another to regulate homeostasis at cell, tissue and organism levels. The mechanisms underlying the introduction of the proper set of disulfide bonds into secretory proteins (oxidative folding) are strictly related to redox homeostasis, ER stress sensing and signaling and provide a good example of the integration systems operative in the early secretory compartment.     

Metabolic signalling in defence and stress: the central roles of soluble redox couples

Plant growth and development are driven by electron transfer reactions. Modifications of redox components are both monitored and induced by cells, and are integral to responses to environmental change. Key redox compounds in the soluble phase of the cell are NAD, NADP, glutathione and ascorbate--all of which interact strongly with reactive oxygen. This review takes an integrated view of the NAD(P)-glutathione-ascorbate network. These compounds are considered not as one-dimensional 'reductants' or 'antioxidants' but as redox couples that can act together to condition cellular redox tone or that can act independently to transmit specific information that tunes signalling pathways. Emphasis is placed on recent developments highlighting the complexity of redox-dependent defence reactions, and the importance of interactions between the reduction state of soluble redox couples and their concentration in mediating dynamic signalling in response to stress. Signalling roles are assessed within the context of interactions with reactive oxygen, phytohormones and calcium, and the biochemical reactions through which redox couples could be sensed are discussed.     

Nuclear glutathione

Glutathione (GSH) is a linchpin of cellular defences in plants and animals with physiologically-important roles in the protection of cells from biotic and abiotic stresses. Moreover, glutathione participates in numerous metabolic and cell signalling processes including protein synthesis and amino acid transport, DNA repair and the control of cell division and cell suicide programmes. While it is has long been appreciated that cellular glutathione homeostasis is regulated by factors such as synthesis, degradation, transport, and redox turnover, relatively little attention has been paid to the influence of the intracellular partitioning on glutathione and its implications for the regulation of cell functions and signalling. We focus here on the functions of glutathione in the nucleus, particularly in relation to physiological processes such as the cell cycle and cell death. The sequestration of GSH in the nucleus of proliferating animal and plant cells suggests that common redox mechanisms exist for DNA regulation in G1 and mitosis in all eukaryotes. We propose that glutathione acts as “redox sensor” at the onset of DNA synthesis with roles in maintaining the nuclear architecture by providing the appropriate redox environment for the DNA replication and safeguarding DNA integrity. In addition, nuclear GSH may be involved in epigenetic phenomena and in the control of nuclear protein degradation by nuclear proteasome. Moreover, by increasing the nuclear GSH pool and reducing disulfide bonds on nuclear proteins at the onset of cell proliferation, an appropriate redox environment is generated for the stimulation of chromatin decompaction. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Redox biology of the intestine

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.