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Cadmium (Cd) is one of the most toxic heavy metals for plants and humans. Reactive oxygen species (ROS) are some of the primary signaling molecules produced after Cd treatment in plants but the contribution of different organelles and specific cell types, together with the impact of light is unknown. We used Arabidopsis lines expressing GRX1-roGFP2 (glutaredoxin1-roGFP) targeted to different cell compartments and analysed changes in redox state over 24 h light/dark cycle in Cd-treated leaf discs. We imaged redox state changes in peroxisomes and chloroplasts in leaf tissue. Chloroplasts and peroxisomes were the most affected organelles in the dark and blocking the photosynthetic electron transport chain (pETC) by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) promotes higher Cd-dependent oxidation in all organelles. Peroxisomes underwent the most rapid changes in redox state in response to Cd and DCMU and silencing chloroplastic NTRC (NADPH thioredoxin reductase C) considerably increases peroxisome oxidation. Total NAD(P)H and cytosolic NADH decreased during exposure to Cd, while Ca+2 content in chloroplasts and cytosol increased in the dark period. Our results demonstrate a Cd-, time- and light-dependent increase of oxidation of all organelles analysed, that could be in part triggered by disturbances in pETC and photorespiration, the decrease of NAD(P)H availability, and differential antioxidants expression at subcellular level.

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Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.

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Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.

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The orientation of membrane proteins within the lipid bilayer is key to understanding their molecular function. Similarly, the proper topology of multispanning membrane proteins is crucial for their function. Although bioinformatics tools can predict these parameters assessing the presence of hydrophobic protein domains sufficiently long to span the membrane and other structural features, the predictions from different algorithms are often inconsistent. Therefore, experimental analysis becomes mandatory. Redox-based topology analysis exploits the steep gradient in the glutathione redox potential (EGSH) across the ER membrane of about 80 mV to visualize the orientation of ER membrane proteins by fusing the EGSH biosensor roGFP2 to either the N- or the C-termini of the investigated protein sequence. Transient expression of these fusion proteins in tobacco leaves allows direct visualization of orientation and topology of ER membrane proteins in planta. The protocol outlined here is based on either a simple merge of the two excitation channels of roGFP2 or a colocalization analysis of the two channels and thus avoids ratiometric analysis of roGFP2 fluorescence.

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Fasting is a popular dietary strategy because it grants numerous advantages, and redox regulation is one mechanism involved. However, the precise redox changes with respect to the redox species, organelles and tissues remain unclear, which hinders the understanding of the metabolic mechanism, and exploring the precision redox map under various dietary statuses is of great significance. Twelve redox-sensitive C. elegans strains stably expressing genetically encoded redox fluorescent probes (Hyperion sensing H2O2 and Grx1-roGFP2 sensing GSH/GSSG) in three organelles (cytoplasm, mitochondria and endoplasmic reticulum (ER)) were constructed in two tissues (body wall muscle and neurons) and were confirmed to respond to redox challenge. The H2O2 and GSSG/GSH redox changes in two tissues and three organelles were obtained by confocal microscopy during fasting, refeeding, and satiation. We found that under fasting condition, H2O2 decreased in most compartments, except for an increase in mitochondria, while GSSG/GSH increased in the cytoplasm of body muscle and the ER of neurons. After refeeding, the redox changes in H2O2 and GSSG/GSH caused by fasting were reversed in most organelles of the body wall muscle and neurons. In the satiated state, H2O2 increased markedly in the cytoplasm, mitochondria and ER of muscle and the ER of neurons, while GSSG/GSH exhibited no change in most organelles of the two tissues except for an increase in the ER of muscle. Our study systematically and precisely presents the redox characteristics under different dietary states in living animals and provides a basis for further investigating the redox mechanism in metabolism and optimizing dietary guidance.

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Hydrogen cyanamide (HC) has been widely used in horticulture to trigger bud burst following dormancy. Its use has been banned in some countries due to human health concerns, however the search for effective safe alternatives is delayed by lack of knowledge of the mechanism of HC action. Earlier studies demonstrate that HC stimulates the production of reactive oxygen species (ROS) and alters the rate of cell division. However, the relationships between HC effects on ROS, redox (reduction/oxidation) homeostasis and cell division are unknown. This study used Arabidopsis thaliana ((L.) Heynh.) seedlings expressing the redox reporter roGFP2 to measure the oxidation states of the nuclei and cytosol in response to HC treatment. The Cytrap dual cell cycle phase marker system and flow cytometry were used to study associated changes in cell proliferation. HC (1.5 mM) reversibly inhibited root growth during a 24 h treatment. Higher concentrations were not reversible. HC did not synchronise the cell cycle, in contrast to hydroxyurea. Rather, HC caused a gradual accumulation of cells in the G2/M phase and decline of G1/S phase cells, 16 to 24 h post-treatment. This was accompanied by increased oxidation of both the nuclei and cytosol. Taken together, these findings show that HC impairs proliferation of embryonic root meristem cells in a reversible manner through restriction of G2/M transition accompanied by increased cellular oxidation.

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Genetically encoded fluorescent biosensors have emerged as a powerful tool to support phenotypic screenings of microbes. Optical analyses of fluorescent sensor signals from colonies grown on solid media can be challenging as imaging devices need to be equipped with appropriate filters matching the properties of fluorescent biosensors. Toward versatile fluorescence analyses of different types of biosensor signals derived from arrayed colonies, we investigate here the use of monochromator equipped microplate readers as an alternative to imaging approaches. Indeed, for analyses of the LacI-controlled expression of the reporter mCherry in Corynebacterium glutamicum, or promoter activity using GFP as reporter in Saccharomyces cerevisiae, an improved sensitivity and dynamic range was observed for a microplate reader-based analyses compared to their analyses via imaging. The microplate reader allowed us to capture signals of ratiometric fluorescent reporter proteins (FRPs) with a high sensitivity and thereby to further improve the analysis of internal pH via the pH-sensitive FRP mCherryEA in Escherichia coli colonies. Applicability of this novel technique was further demonstrated by assessing redox states in C. glutamicum colonies using the FRP Mrx1-roGFP2. By the use of a microplate reader, oxidative redox shifts were measured in a mutant strain lacking the non-enzymatic antioxidant mycothiol (MSH), indicating its major role for maintaining a reduced redox state also in colonies on agar plates. Taken together, analyses of biosensor signals from microbial colonies using a microplate reader allows comprehensive phenotypic screenings and thus facilitates further development of new strains for metabolic engineering and systems biology.

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Hydrogen peroxide (H2O2) is an important signaling molecule involved in regulating antioxidative transcriptional responses, cellular differentiation, and hypoxia response. H2O2 generation and signaling are highly localized processes. Understanding the dynamics of this molecule inside intact cells with subcompartmental resolution is instrumental to unravel its role in cellular signaling. Different genetically encoded fluorescent sensors have been developed over the last few years that enable such non-disruptive monitoring with high spatiotemporal resolution. In this chapter, we describe the use of these genetically encoded sensors to directly monitor H2O2 dynamics in yeast and cultured mammalian cells.

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Late blight caused by the oomycete Phytophthora infestans is a most devastating disease of potatoes (Solanum tuberosum). Its early detection is crucial for suppressing disease spread. Necrotic lesions are normally seen in leaves at 4 days post-inoculation (dpi) when colonized cells are dead, but early detection of the initial biotrophic growth stage, when the pathogen feeds on living cells, is challenging. Here, the biotrophic growth phase of P. infestans was detected by whole-plant redox imaging of potato plants expressing chloroplast-targeted reduction-oxidation sensitive green fluorescent protein (chl-roGFP2). Clear spots on potato leaves with a lower chl-roGFP2 oxidation state were detected as early as 2 dpi, before any visual symptoms were recorded. These spots were particularly evident during light-to-dark transitions, and reflected the mislocalization of chl-roGFP2 outside the chloroplasts. Image analysis based on machine learning enabled systematic identification and quantification of spots, and unbiased classification of infected and uninfected leaves in inoculated plants. Comparing redox with chlorophyll fluorescence imaging showed that infected leaf areas that exhibit mislocalized chl-roGFP2 also showed reduced non-photochemical quenching and enhanced quantum PSII yield (ΦPSII) compared with the surrounding leaf areas. The data suggest that mislocalization of chloroplast-targeted proteins is an efficient marker of late blight infection, and demonstrate how it can be utilized for non-destructive monitoring of the disease biotrophic stage using whole-plant redox imaging.

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The redox state of the host-parasite unit has been hypothesized to play a central role for the fitness of the intraerythrocytic blood stages of the human malaria parasite Plasmodium falciparum. In particular, hemoglobinopathies have been suggested to cause a more oxidizing environment, thereby protecting from severe malaria. Here we determined the redox potential of infected wild-type (hemoglobin AA) or sickle trait (hemoglobin AS) erythrocytes using parasite-encoded variants of the redox-sensitive green-fluorescent protein 2 (roGFP2). Our non-invasive roGFP2 single-cell measurements revealed a reducing steady-state redox potential of -304 ± 11 mV for the erythrocyte cytosol during ring-stage development and a rather sudden oxidation to -278 ± 12 mV during trophozoite-stage development around 28 h post invasion. There was no significant difference between wild-type or sickle trait erythrocytes regarding the stage dependence and the detected increase of the redox potential during the intraerythrocytic life cycle. The steady-state redox potential of the parasite cytosol, between -304 and -313 mV, was highly reducing throughout the life cycle. The redox potential in the parasitophorous vacuole at the interface between the secretory pathway and the erythrocyte was -284 ± 10 mV and remained stable during trophozoite-stage development with implications for the export of disulfide-containing proteins. In summary, P. falciparum blood stage development from the late ring to the early trophozoite stage causes a physiological jump in erythrocyte redox potential irrespective of the presence or absence of hemoglobin S.

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Reactive oxygen species (ROS) serve as second messengers in plant signaling pathways to remodel plant growth and development. New insights into how enzymatic ROS-producing machinery is regulated by hormones or localized during development have provided a framework for understanding the mechanisms that control ROS accumulation patterns. Signaling-mediated increases in ROS can then modulate the activity of proteins through reversible oxidative modification of specific cysteine residues. Plants also control the synthesis of antioxidants, including plant-specialized metabolites, to further define when, where, and how much ROS accumulate. The availability of sophisticated imaging capabilities, combined with a growing tool kit of ROS detection technologies, particularly genetically encoded biosensors, sets the stage for improved understanding of ROS as signaling molecules.

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Selenium is an essential trace element required for seleno-protein synthesis in many eukaryotic cells excluding higher plants. However, a substantial fraction of organically bound selenide in human nutrition is directly or indirectly derived from plants, which assimilate inorganic selenium into organic seleno-compounds. In humans, selenium deficiency is associated with several health disorders Despite its importance for human health, selenium assimilation and metabolism is barely understood in plants. Here, we analyzed the impact of the two dominant forms of soil-available selenium, selenite and selenate, on plant development and selenium partitioning in plants. We found that the reference plant Arabidopsis thaliana discriminated between selenate and selenite application. In contrast to selenite, selenate was predominantly deposited in leaves. This explicit deposition of selenate caused chlorosis and impaired plant morphology, which was not observed upon selenite application. However, only selenate triggered the accumulation of the macronutrient sulfur, the sister element of selenium in the oxygen group. To understand the oxidation state-specific toxicity mechanisms for selenium in plants, we quantified the impact of selenate and selenite on the redox environment in the plastids and the cytosol in a time-resolved manner. Surprisingly, we found that selenite first caused the oxidation of the plastid-localized glutathione pool and had a marginal impact on the redox state of the cytosolic glutathione pool, specifically in roots. In contrast, selenate application caused more vigorous oxidation of the cytosolic glutathione pool but also impaired the plastidic redox environment. In agreement with the predominant deposition in leaves, the selenate-induced oxidation of both glutathione pools was more pronounced in leaves than in roots. Our results demonstrate that Se-species dependent differences in Se partitioning substantially contribute to whole plant Se toxicity and that these Se species have subcellular compartment-specific impacts on the glutathione redox buffer that correlate with toxicity symptoms.

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Plant cells produce reactive oxygen species (ROS) as by-products of oxygen metabolism and for signal transduction. Depending on their concentration and their site of production, ROS can cause oxidative damage within the cell and must be effectively scavenged. Detoxification of the most stable ROS, hydrogen peroxide (H2O2), via the glutathione-ascorbate pathway may transiently alter the glutathione redox potential (EGSH). Changes in EGSH can thus be considered as an indicator of the oxidative load in the cell. Genetically encoded probes based on roGFP2 enable extended opportunities for in vivo monitoring of H2O2 and EGSH dynamics. Here, we provide detailed protocols for live monitoring of both parameters in the cytosol with the probes Grx1-roGFP2 for EGSH and roGFP2-Orp1 for H2O2, respectively. The protocols have been adapted for live cell imaging with high lateral resolution on a confocal microscope and for multi-parallel measurements in whole organs or intact seedlings in a fluorescence microplate reader. Elicitor-induced ROS generation is used for illustration of the opportunities for dynamic ROS measurements that can be transferred to other research questions and model systems.

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Mitochondria are complex organelles with multifaceted roles in cell biology, acting as signaling hubs that implicate them in cellular physiology and pathology. Mitochondria are both the target and the origin of multiple signaling events, including redox processes and calcium signaling which are important for organellar function and homeostasis. One way to interrogate mitochondrial function is by live cell imaging. Elaborated approaches perform imaging of single mitochondrial dynamics in living cells and animals. Imaging mitochondrial signaling and function can be challenging due to the sheer number of mitochondria, and the speed, propagation, and potential short half-life of signals. Moreover, mitochondria are organized in functionally coupled interorganellar networks. Therefore, advanced analysis and postprocessing tools are needed to enable automated analysis to fully quantitate mitochondrial signaling events and decipher their complex spatiotemporal connectedness. Herein, we present a protocol for recording and automating analyses of signaling in neuronal mitochondrial networks.

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Cell metabolism heavily relies on the redox reactions that inevitably generate reactive oxygen species (ROS). It is now well established that ROS fluctuations near basal levels coordinate numerous physiological processes in living organisms, thus exhibiting regulatory functions. Hydrogen peroxide, the most long-lived ROS, is a key contributor to ROS-dependent signal transduction in the cell. H2 O2 is known to impact various targets in the cell; therefore, the question of how H2 O2 modulates physiological processes in a highly specific manner is central in redox biology. To resolve this question, novel genetic tools have recently been created for detecting H2 O2 and emulating its generation in living organisms with unmatched spatiotemporal resolution. Here, we review H2 O2 -sensitive genetically encoded fluorescent sensors and opto- and chemogenetic tools for controlled H2 O2 generation.

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Background Heart failure is responsible for approximately 65% of deaths in patients with type 2 diabetes mellitus. However, existing therapeutics for type 2 diabetes mellitus have limited success on the prevention of diabetic cardiomyopathy. The aim of this study was to determine whether moderate elevation in D-ß-hydroxybutyrate improves cardiac function in animals with type 2 diabetes mellitus. Methods and Results Type 2 diabetic (db/db) and their corresponding wild-type mice were fed a control diet or a diet where carbohydrates were equicalorically replaced by D-ß-hydroxybutyrate-(R)-1,3 butanediol monoester (ketone ester [KE]). After 4 weeks, echocardiography demonstrated that a KE diet improved systolic and diastolic function in db/db mice. A KE diet increased expression of mitochondrial succinyl-CoA:3-oxoacid-CoA transferase and restored decreased expression of mitochondrial ß-hydroxybutyrate dehydrogenase, key enzymes in cardiac ketone metabolism. A KE diet significantly enhanced both basal and ADP-mediated oxygen consumption in cardiac mitochondria from both wild-type and db/db animals; however, it did not result in the increased mitochondrial respiratory control ratio. Additionally, db/db mice on a KE diet had increased resistance to oxidative and redox stress, with evidence of restoration of decreased expression of thioredoxin and glutathione peroxidase 4 and less permeability transition pore activity in mitochondria. Mitochondrial biogenesis, quality control, and elimination of dysfunctional mitochondria via mitophagy were significantly increased in cardiomyocytes from db/db mice on a KE diet. The increase in mitophagy was correlated with restoration of mitofusin 2 expression, which contributed to improved coupling between cytosolic E3 ubiquitin ligase translocation into mitochondria and microtubule-associated protein 1 light chain 3-mediated autophagosome formation. Conclusions Moderate elevation in circulating D-ß-hydroxybutyrate levels via KE supplementation enhances mitochondrial biogenesis, quality control, and oxygen consumption and increases resistance to oxidative/redox stress and mPTP opening, thus resulting in improvement of cardiac function in animals with type 2 diabetes mellitus.

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The thioredoxin fold superfamily is highly diverse and contains many enzymatically active glutathione-dependent thiol-disulfide oxidoreductases, for example glutaredoxins and protein disulfide isomerases. However, many thioredoxin fold proteins remain completely uncharacterized, their cellular function is unknown, and it is unclear if they have a redox-dependent enzymatic activity with glutathione or not. Investigation of enzymatic activity traditionally involved time-consuming in vitro characterization of recombinant proteins, limiting the capacity to study novel mechanisms and structure-function relationships. To accelerate our investigation of glutathione-dependent oxidoreductases, we have developed a high-throughput and semi-quantitative assay in yeast. We combined overexpression of the glutathione transporter OPT1 with genetic fusion constructs between glutathione-dependent oxidoreductases and redox-sensitive green fluorescent protein 2 (roGFP2) to allow the rapid characterization of enzymatic activity with physiological substrates. We show that the kinetics of roGFP2 oxidation by glutathione disulfide correlate well with the in vitro-determined activity of the genetically fused glutaredoxins or mutants thereof. Our assay thus allows direct screening of glutaredoxin activity and rapid investigation of structure-function relationships. We also demonstrate that our assay can be used to monitor roGFP2 oxidation by S-nitrosoglutathione (GSNO). We show that glutaredoxins efficiently catalyze oxidation of roGFP2 by GSNO in both live yeast cells and in vitro. In summary, we have established a novel assay for activity screening and characterization of glutathione-dependent oxidoreductases.

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Genetically encoded fluorescent H2O2 probes continue to advance the field of redox biology. Here, we compare the previously established peroxiredoxin-based H2O2 probe roGFP2-Tsa2ΔCR with the newly described OxyR-based H2O2 probe HyPer7, using yeast as the model system. Although not as sensitive as roGFP2-Tsa2ΔCR, HyPer7 is much improved relative to earlier HyPer versions, most notably by ratiometric pH stability. The most striking difference between the two probes is the dynamics of intracellular probe reduction. HyPer7 is rapidly reduced, predominantly by the thioredoxin system, whereas roGFP2-Tsa2ΔCR is reduced more slowly, predominantly by the glutathione system. We discuss the pros and cons of each probe and suggest that future side-by-side measurements with both probes may provide information on the relative activity of the two major cellular reducing systems.

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The green peach aphid (Myzus persicae) is a phloem-feeding insect that causes economic damage on a wide array of crops. Using a luminol-based assay, a superoxide-responsive reporter gene (Zat12::luciferase), and a probe specific to hydrogen peroxide (HyPer), we demonstrated that this aphid induces accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. Similar to the apoplastic oxidative burst induced by pathogens, this response to aphids was rapid and transient, with two peaks occurring within 1 and 4 hr after infestation. Aphid infestation also induced an oxidative response in the cytosol and peroxisomes, as measured using a redox-sensitive variant of green fluorescent protein (roGFP2). This intracellular response began within minutes of infestation but persisted 20 hr or more after inoculation, and the response of the peroxisomes appeared stronger than the response in the cytosol. Our results suggest that the oxidative response to aphids involves both apoplastic and intracellular sources of ROS, including ROS generation in the peroxisomes, and these different sources of ROS may potentially differ in their impacts on host suitability for aphids.

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H2S-producing enzymes in bacteria have been shown to be closely engaged in the process of microbial survival and antibiotic resistance. However, no inhibitors have been discovered for these enzymes, e.g., 3-mercaptopyruvate sulfurtransferase (MST). In the present study, we identified several classes of inhibitors for Escherichia coli MST (eMST) through high-throughput screening of ∼26,000 compounds. The thiazolidinedione-type inhibitors were found to selectively bind to Arg178 and Ser239 residues of eMST but hardly affected human MST. Moreover, the pioglitazone of this class concentration dependently accumulates the 3-mercaptopyruvate substrate and suppresses the H2S and reactive sulfane sulfur products in bacteria. Importantly, pioglitazone could potentiate the level of reactive oxygen species in cellulo and consequently enhance the antimicrobial effects of gentamicin and macrophages in culture. This study has identified the bioactive inhibitor of eMST, paving the way for the pharmacological targeting of eMST to synergistically control the survival of E. coli.

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