Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification.

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.

Early detection of late blight in potato by whole-plant redox imaging.

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.

A guide to genetically-encoded redox biosensors: State of the art and opportunities.

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.

A comparison of Prx- and OxyR-based H2O2 probes expressed in S. cerevisiae.

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.

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1.

Deoxynyboquinone (DNQ), a potent novel quinone-based antineoplastic agent, selectively kills solid cancers with overexpressed cytosolic NAD(P)H:quinone oxidoreductase-1 (NQO1) via excessive ROS production. A genetically encoded redox-sensitive probe was used to monitor intraorganellar glutathione redox potentials (EGSH) as a direct indicator of cellular oxidative stress following chemotherapeutic administration. Beta-lapachone (ß-lap) and DNQ-induced spatiotemporal redox responses were monitored in human lung A549 and pancreatic MIA-PaCa-2 adenocarcinoma cells incubated with or without dicumarol and ES936, potent NQO1 inhibitors. Immediate oxidation of EGSH in both the cytosol and mitochondrial matrix was observed in response to DNQ and ß-lap. The DNQ-induced cytosolic oxidation was fully prevented with NQO1 inhibition, whereas mitochondrial oxidation in A549 was NQO1-independent in contrast to MIA-PaCa-2 cells. However, at pharmacologic concentrations of ß-lap both quinone-based substrates directly oxidized the redox probe, a possible sign of off-target reactivity with cellular thiols. Together, these data provide new evidence that DNQ's direct and discerning NQO1 substrate specificity underlies its pharmacologic potency, while ß-lap elicits off-target responses at its effective doses.

Glutathione peroxidase-like enzymes cover five distinct cell compartments and membrane surfaces in Arabidopsis thaliana.

Glutathione peroxidase-like enzymes (GPXLs) constitute a family of eight peroxidases in Arabidopsis thaliana. In contrast to the eponymous selenocysteine glutathione peroxidases in mammalian cells that use glutathione as electron donor, GPXLs rely on cysteine instead of selenocysteine for activity and depend on the thioredoxin system for reduction. Although plant GPXLs have been implicated in important agronomic traits such as drought tolerance, photooxidative tolerance and immune responses, there remain major ambiguities regarding their subcellular localization. Because their site of action is a prerequisite for an understanding of their function, we investigated the localization of all eight GPXLs in stable Arabidopsis lines expressing N-terminal and C-terminal fusions with redox-sensitive green fluorescent protein 2 (roGFP2) using confocal microscopy. GPXL1 and GPXL7 were found in plastids, while GPXL2 and GPXL8 are cytosolic nuclear. The N-terminal target peptide of GPXL6 is sufficient to direct roGFP2 into mitochondria. Interestingly, GPXL3, GPXL4 and GPXL5 all appear to be membrane bound. GPXL3 was found exclusively in the secretory pathway where it is anchored by a single N-terminal transmembrane domain. GPXL4 and GPXL5 are anchored to the plasma membrane. Presence of an N-terminal myristoylation motif and genetic disruption of membrane association through targeted mutagenesis point to myristoylation as essential for membrane localization.

Chasing stress signals - Exposure to extracellular stimuli differentially affects the redox state of cell compartments in the wild type and signaling mutants of Botrytis cinerea.

Reactive oxygen species (ROS) are important molecules influencing intracellular developmental processes as well as plant pathogen interactions. They are produced at the infection site and affect the intracellular redox homeostasis. However, knowledge of ROS signaling pathways, their connection to other signaling cascades, and tools for the visualization of intra- and extracellular ROS levels and their impact on the redox state are scarce. By using the genetically encoded biosensor roGFP2 we studied for the first time the differences between the redox states of the cytosol, the intermembrane space of mitochondria and the ER in the filamentous fungus Botrytis cinerea. We showed that the ratio of oxidized to reduced glutathione inside of the cellular compartments differ and that the addition of hydrogen peroxide (H2O2), calcium chloride (CaCl2) and the fluorescent dye calcofluor white (CFW) have a direct impact on the cellular redox states. Dependent on the type of stress agents applied, the redox states were affected in the different cellular compartments in a temporally shifted manner. By integrating the biosensor in deletion mutants of bcnoxA, bcnoxB, bctrx1 and bcltf1 we further elucidated the putative roles of the different proteins in distinct stress-response pathways. We showed that the redox states of ΔbcnoxA and ΔbcnoxB display a wild-type pattern upon exposure to H2O2, but appear to be strongly affected by CaCl2 and CFW. Moreover, we demonstrated the involvement of the light-responsive transcription factor BcLtf1 in the maintenance of the redox state in the intermembrane space of the mitochondria. Finally, we report that CaCl2 as well as cell wall stress-inducing agents stimulate ROS production and that ΔbcnoxB produces significantly less ROS than the wild type and ΔbcnoxA.

Robust anti-oxidant defences in the rice blast fungus Magnaporthe oryzae confer tolerance to the host oxidative burst.

Plants respond to pathogen attack via a rapid burst of reactive oxygen species (ROS). However, ROS are also produced by fungal metabolism and are required for the development of infection structures in Magnaporthe oryzae. To obtain a better understanding of redox regulation in M. oryzae, we measured the amount and redox potential of glutathione (E(GSH)), as the major cytoplasmic anti-oxidant, the rates of ROS production, and mitochondrial activity using multi-channel four-dimensional (x,y,z,t) confocal imaging of Grx1-roGFP2 and fluorescent reporters during spore germination, appressorium formation and infection. High levels of mitochondrial activity and ROS were localized to the growing germ tube and appressorium, but E(GSH) was highly reduced and tightly regulated during development. Furthermore, germlings were extremely resistant to external H2O2 exposure ex planta. EGSH remained highly reduced during successful infection of the susceptible rice cultivar CO39. By contrast, there was a dramatic reduction in the infection of resistant (IR68) rice, but the sparse hyphae that did form also maintained a similar reduced E(GSH). We conclude that M. oryzae has a robust anti-oxidant defence system and maintains tight control of EGSH despite substantial oxidative challenge. Furthermore, the magnitude of the host oxidative burst alone does not stress the pathogen sufficiently to prevent infection in this pathosystem.

The yeast oligopeptide transporter Opt2 is localized to peroxisomes and affects glutathione redox homeostasis.

Glutathione, the most abundant small-molecule thiol in eukaryotic cells, is synthesized de novo solely in the cytosol and must subsequently be transported to other cellular compartments. The mechanisms of glutathione transport into and out of organelles remain largely unclear. We show that budding yeast Opt2, a close homolog of the plasma membrane glutathione transporter Opt1, localizes to peroxisomes. We demonstrate that deletion of OPT2 leads to major defects in maintaining peroxisomal, mitochondrial, and cytosolic glutathione redox homeostasis. Furthermore, ∆opt2 strains display synthetic lethality with deletions of genes central to iron homeostasis that require mitochondrial glutathione redox homeostasis. Our results shed new light on the importance of peroxisomes in cellular glutathione homeostasis.

Visualizing Orientation and Topology of ER Membrane Proteins In Planta.

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.

Cadmium exposure induced light/dark- and time-dependent redox changes at subcellular level in Arabidopsis plants.

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.

Hydrogen Cyanamide Causes Reversible G2/M Cell Cycle Arrest Accompanied by Oxidation of the Nucleus and Cytosol.

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.

Real-Time Monitoring of Hydrogen Peroxide Levels in Yeast and Mammalian Cells.

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.

Exploring the precision redox map during fasting-refeeding and satiation in C. elegans.

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.

Precise and versatile microplate reader-based analyses of biosensor signals from arrayed microbial colonies.

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.

Discriminative Long-Distance Transport of Selenate and Selenite Triggers Glutathione Oxidation in Specific Subcellular Compartments of Root and Shoot Cells in Arabidopsis.

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.

Physiological jump in erythrocyte redox potential during Plasmodium falciparum development occurs independent of the sickle cell trait.

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.

Live Monitoring of ROS-Induced Cytosolic Redox Changes with roGFP2-Based Sensors in Plants.

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.

Automated Quantification and Network Analysis of Redox Dynamics in Neuronal Mitochondria.

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.

A guide to genetically encoded tools for the study of H2 O2.

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.