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Aims: Genetically encoded GFP-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2). Results: SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity in cellulo. SfroGFP2-based redox biosensors are pH-insensitive in a physiological pH range and show midpoint potentials comparable to roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved fluorescence intensity in cellulo. Innovation: This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited. Conclusion: Improved structural stability of sfroGFP2 gives rise to increased fluorescence intensity in cellulo. Fusion to hGrx1 provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general.

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers such as the highly reduced glutathione pool serve as reservoirs of reducing power for several ROS scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide (GSSG) and a shift of the glutathione redox potential (EGSH) towards less negative values is considered a hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis seedlings and recorded dynamic changes in EGSH and H2O2 levels with the genetically-encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) targeted to chloroplasts, the cytosol or mitochondria. Treatment of seedlings with MV caused a rapid oxidation in chloroplasts and subsequently also in the cytosol and mitochondria. The MV-induced oxidation was significantly boosted by illumination with actinic light and largely abolished by inhibitors of photosynthetic electron transport. In addition, MV also induced an autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress acclimation in plants.One sentence summaryMethyl viologen-induced photooxidative stress causes an increase of H2O2 and oxidation of glutathione in chloroplasts, cytosol and mitochondria as well as autonomous oxidation in mitochondria.

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (EGSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in EGSH and H2O2 levels with the genetically encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

The new phytologist 224(4), 1668-1684 (2019). doi:10.1111/nph.16093, Published by Wiley-Blackwell, Oxford [u.a.]

Hydrogen peroxide (H2 O2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2 O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H2 O2 sensing in living plants. We established H2 O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H2 O2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2 O2 , pharmacologically-induced H2 O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2 O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants. We provided a well defined toolkit for H2 O2 monitoring in planta and showed that intracellular H2 O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2 O2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.

Hydrogen sulfide (H2S) is an important gaseous signaling molecule in plants that participates in stress responses and development. L-Cys desulfhydrase 1, one of the enzymatic sources of H2S in plants, participates in abscisic acid-induced stomatal closure. We combined pharmacological and genetic approaches to elucidate the involvement of H2S in stomatal closure and the interplay between H2S and other second messengers of the guard cell signaling network, such as hydrogen peroxide (H2O2) and phospholipase D (PLD)-derived phosphatidic acid in Arabidopsis (Arabidopsis thaliana). Both NADPH oxidase isoforms, respiratory burst oxidase homolog (RBOH)D and RBOHF, were required for H2S-induced stomatal closure. In vivo imaging using the cytosolic ratiometric fluorescent biosensor roGFP2-Orp1 revealed that H2S stimulates H2O2 production in Arabidopsis guard cells. Additionally, we observed an interplay between H2S and PLD activity in the regulation of reactive oxygen species production and stomatal movement. The PLDα1 and PLDδ isoforms were required for H2S-induced stomatal closure, and most of the H2S-dependent H2O2 production required the activity of PLDα1. Finally, we showed that H2S induced increases in the PLDδ-derived phosphatidic acid levels in guard cells. Our results revealed the involvement of H2S in the signaling network that controls stomatal closure, and suggest that H2S regulates NADPH oxidase and PLD activity in guard cells. Fil: Scuffi, Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Nietzel, Thomas. Westfalische Wilhelms Universitat; Alemania. Universitat Bonn; Alemania Fil: Di Fino, Luciano Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Meyer, Andreas J.. Universitat Bonn; Alemania Fil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Schwarzlände, Markus. Westfalische Wilhelms Universitat; Alemania. Universitat Bonn; Alemania Fil: Laxalt, Ana Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina Fil: Garcia-Mata, Carlos. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentina

Significance By studying in vivo changes of ATP levels in the plastids and cytosol of Arabidopsis thaliana using a FRET-based ATP sensor, we show that the plastidic ATP concentrations in cotyledon, hypocotyl, and root of 10-day-old seedlings are significantly lower than the cytosolic concentrations. We show that chloroplasts consume ATP rapidly and the import of ATP into mature chloroplasts is impeded by the low density of NTT transporter. Hence, unlike in diatoms, where ATP is imported into chloroplasts to support the Calvin–Benson–Bassham (CBB) cycle, mature chloroplasts of Arabidopsis do not balance the ATP:NADPH ratio by importing ATP from the cytosol. Rather, chloroplasts can export surplus reducing equivalents, which can be used by the mitochondria to supply ATP to the cytosol., Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin–Benson–Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2− concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here we establish live MgATP2−assessment in plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensorin vitrounder conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2−changesin planta. A MgATP2−map of the Arabidopsis seedling highlights different MgATP2−concentrations between tissues and in individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.One-sentence SummarySensing of MgATP2−by fluorimetry and microscopy allows dissection of ATP fluxes of isolated organelles, and dynamics of cytosolic MgATP2−in vivo.Funding AgenciesThis work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Emmy-Noether programme (SCHW1719/1-1; M.S. and GR4251/1-1; C.G.), the Research Training Group GRK 2064 (M.S.; A.J.M.), the Priority Program SPP1710 (A.J.M.) and a grant (SCHW1719/5-1; M.S.) as part of the package PAK918. The Seed Fund grant CoSens from the Bioeconomy Science Center, NRW (A.J.M.; M.S.) is gratefully acknowledged. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-002 13). A.Co. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca through the FIRB 2010 programme (RBFR10S1LJ_001) and Piano di Sviluppo di Ateneo 2015 (Università degli Studi di Milano). M.Z. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca (Italy) through the PRIN 2010 programme (PRIN2010CSJX4F). S.W. and T.N. received travel support by the Deutscher Akademischer Austauschdienst (DAAD). V.D.C. was supported by the European Social Fund, Operational Programme 2007/2013, and an Erasmus+ Traineeship grant. M.D.F was supported by The Human Frontier Science Program (RPG0053/2012), and the Leverhulme Foundation (RPG-2015-437). I.M.M. was supported by a grant from the Danish Council for Independent Research - Natural Sciences. V.C.P. was supported by the Innovation and Technology Fund (Funding Support to Partner State Key Laboratories in Hong Kong) of the HKSAR.AbbreviationsAAC – ADP/ATP carrier; AK – adenylate kinase; cAT – carboxyatractyloside; CCCP – carbonyl cyanide m-chlorophenyl hydrazone; CFP – cyan fluorescent protein; CLSM – confocal laser scanning microscopy; ETC – electron transport chain; FRET – Förster Resonance Energy Transfer; LSFM – light sheet fluorescence microscopy.

Globulins are an important group of seed storage proteins in dicotyledonous plants. They are synthesized during seed development, assembled into very compact protein complexes, and finally stored in protein storage vacuoles (PSVs). Here, we report a proteomic investigation on the native composition and structure of cruciferin, the 12 S globulin of Brassica napus. PSVs were directly purified from mature seeds by differential centrifugations. Upon analyses by blue native (BN) PAGE, two major types of cruciferin complexes of ∼ 300–390 kDa and of ∼470 kDa are resolved. Analyses by two-dimensional BN/SDS-PAGE revealed that both types of complexes are composed of several copies of the cruciferin α and β polypeptide chains, which are present in various isoforms. Protein analyses by two-dimensional isoelectric focusing (IEF)/SDS-PAGE not only revealed different α and β isoforms but also several further versions of the two polypeptide chains that most likely differ with respect to posttranslational modifications. Overall, more than 30 distinct forms of cruciferin were identified by mass spectrometry. To obtain insights into the structure of the cruciferin holocomplex, a native PSV fraction was analyzed by single particle electron microscopy. More than 20,000 images were collected, classified, and used for the calculation of detailed projection maps of the complex. In contrast to previous reports on globulin structure in other plant species, the cruciferin complex of Brassica napus has an octameric barrel-like structure, which represents a very compact building block optimized for maximal storage of amino acids within minimal space. Background: Cruciferin represents the most abundant protein in Brassica napus seeds where its efficient storage is essential under minimized space conditions. Results: The cruciferin complex has an octameric barrel-like structure of ∼420 kDa. Conclusion: The barrel-like structure represents a compact building block optimized for maximal storage of amino acids. Significance: Novel insights into structure and packing of seed storage proteins.

Mitochondria are hotspots of cellular redox biochemistry. Respiration as a defining mitochondrial function is made up of a series of electron transfers that are ultimately coupled to maintaining the proton motive force, ATP production and cellular energy supply. The individual reaction steps involved require tight control and flexible regulation to maintain energy and redox balance in the cell under fluctuating demands. Redox regulation by thiol switching has been a long-standing candidate mechanism to support rapid adjustment of mitochondrial protein function at the posttranslational level. Here we review recent advances in our understanding of cysteine thiol switches in the mitochondrial proteome with a focus on their operation in vivo. We assess the conceptual basis for thiol switching in mitochondria and discuss to what extent insights gained from in vitro studies may be valid in vivo, considering thermodynamic, kinetic and structural constraints. We compare functional proteomic approaches that have been used to assess mitochondrial protein thiol switches, including thioredoxin trapping, redox difference gel electrophoresis (redoxDIGE), isotope-coded affinity tag (OxICAT) and iodoacetyl tandem mass tag (iodoTMT) labelling strategies. We discuss conditions that may favour active thiol switching in mitochondrial proteomes in vivo, and appraise recent advances in dissecting their impact using combinations of in vivo redox sensing and quantitative redox proteomics. Finally we focus on four central facets of mitochondrial biology, aging, carbon metabolism, energy coupling and electron transport, exemplifying the current emergence of a mechanistic understanding of mitochondrial regulation by thiol switching in living plants and animals.

Mitochondrial physiology sets the basis for function of the organelle and vice versa. While a limited range of in vivo parameters, such as oxygen consumption, has been classically accessible for measurement, a growing collection of fluorescent protein sensors can now give insights into the physiology of plant mitochondria. Nevertheless, the meaningful application of these sensors in mitochondria is technically challenging and requires rigorous experimental standards. Here we exemplify the application of three genetically encoded sensors to monitor glutathione redox potential, pH, and calcium in the matrix of mitochondria in intact plants. We describe current methods for quantitative imaging and analysis in living root tips by confocal microscopy and discuss methodological limitations.

The iron-sulfur cluster (ISC) is an ancient and essential cofactor of many proteins involved in electron transfer and metabolic reactions. In Arabidopsis, three pathways exist for the maturation of iron-sulfur proteins in the cytosol, plastids, and mitochondria. We functionally characterized the role of mitochondrial glutaredoxin S15 (GRXS15) in biogenesis of ISC containing aconitase through a combination of genetic, physiological, and biochemical approaches. Two Arabidopsis T-DNA insertion mutants were identified as null mutants with early embryonic lethal phenotypes that could be rescued by GRXS15. Furthermore, we showed that recombinant GRXS15 is able to coordinate and transfer an ISC and that this coordination depends on reduced glutathione (GSH). We found the Arabidopsis GRXS15 able to complement growth defects based on disturbed ISC protein assembly of a yeast Delta grx5 mutant. Modeling of GRXS15 onto the crystal structures of related nonplant proteins highlighted amino acid residues that after mutation diminished GSH and subsequently ISC coordination, as well as the ability to rescue the yeast mutant. When used for plant complementation, one of these mutant variants, GRXS15(K83/A), led to severe developmental delay and a pronounced decrease in aconitase activity by approximately 65%. These results indicate that mitochondrial GRXS15 is an essential protein in Arabidopsis, required for full activity of iron-sulfur proteins.

Mitochondrial physiology sets the basis for function of the organelle and vice versa. While a limited range of in vivo parameters, such as oxygen consumption, has been classically accessible for measurement, a growing collection of fluorescent protein sensors can now give insights into the physiology of plant mitochondria. Nevertheless, the meaningful application of these sensors in mitochondria is technically challenging and requires rigorous experimental standards. Here we exemplify the application of three genetically encoded sensors to monitor glutathione redox potential, pH, and calcium in the matrix of mitochondria in intact plants. We describe current methods for quantitative imaging and analysis in living root tips by confocal microscopy and discuss methodological limitations.