The spatiotemporal organization of proteins and lipids within membranes is crucial for ensuring proper cellular signaling. While the segregation of proteins and lipids into membrane nanodomains is well established, it remains unclear whether nanodomains can generate gradients of small diffusible molecules. In plants, reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), act as key signaling molecules in response to environmental stimuli such as osmotic stress. However, how extracellular H2O2 affects intracellular signaling has remained unknown. Here, we show that osmotic stimulation induces the formation of localized, H2O2-rich nanoenvironments at the cytoplasmic face of the plasma membrane (PM) in Arabidopsis root cells. Using a PM-tethered H2O2 biosensor, we found that these oxidized nanodomains arise from the clustering of RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs) and RHO OF PLANTS 6 (ROP6), in coordination with aquaporin-mediated H2O2 transport via the PLASMA MEMBRANE INTRINSIC PROTEIN2;7 (PIP2;7). These local redox hotspots at the PM create a feedforward loop in which H2O2 enhances ROP6 nanoclustering, thereby amplifying ROS signaling. Disruption of H2O2 production or transport dampens both ROP6 clustering and anisotropic cell expansion, indicating a crucial role for spatially confined redox signaling in regulating plant growth under osmotic stress. Our findings propose a model in which ROP6/RBOHD-F/PIP2;7 nanodomains function as discrete redox signaling units, redefining ROS signaling at the PM as a structured, signal-specific, and compartmentalized process.

The metabolic flexibility of plants enables them to cope particularly well with changing environmental conditions. This flexibility is achieved by cellular processes that require tight coordination in space and time and constant balancing to maximise plant fitness. If we want to identify crops with higher yields and improved resistance to abiotic and biotic stresses, then we need to unravel these metabolic processes experimentally, and genetically encoded biosensors (GEBs) seem ideal for this. They allow non-invasive monitoring of metabolic processes in living cells over time and with high spatial and temporal resolution. The list of sensors and sensor variants that have been developed or established in plants continues to grow, providing insights into more and more parameters of plant metabolism. This, together with technological advances, also facilitates paraplexing and multiplexing experiments, where several processes are monitored simultaneously by GEBs. Despite these advantages, GEBs need to be used carefully and users must fully understand their characteristics in the chosen experimental plant system in order to draw meaningful conclusions from the spectroscopic changes of a sensor. Here, we aim to provide a list of fluorescent GEBs that can be selected for in planta use and highlight recent biological insights gained from them, focusing on advances where multiple GEBs have been used. We also discuss criteria for selecting an appropriate sensor and aspects of the field that remain challenging, in the hope of helping plant scientists to generate and interpret plant metabolism data using GEBs in a meaningful way.

Photosynthesis provides energy and organic substrates to most life. In plants, photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects energy physiology within and beyond the chloroplast. We developed a new standard for the live-monitoring of subcellular energy physiology by combining confocal imaging of genetically encoded fluorescent protein biosensors with advanced on-stage illumination technology to investigate pH, MgATP[2][1]- and NADH/NAD+ dynamics at dark-light transitions in Arabidopsis mesophyll cells. Our findings reveal a stromal alkalinization signature induced by photosynthetic proton pumping, extending to the cytosol and mitochondria as an ’alkalinization wave’. Photosynthesis leads to increased MgATP[2][1]- levels in both the stroma and cytosol. Additionally, we observed reduction of the NAD pool driven by photosynthesis-derived electron export. Arabidopsis lines defective in chloroplast NADP- and mitochondrial NAD-dependent malate dehydrogenases show more reduced cytosolic NAD redox status even in darkness, highlighting the involvement of chloroplasts and mitochondria in shaping cytosolic redox metabolism via malate metabolism. Our study sets a novel methodological standard for precision live-monitoring of photosynthetic cell physiology. Applying this technology reveals signatures of photosynthetic physiology within and beyond the chloroplast with unprecedented resolution. Those signatures link photosynthetic activity and the fundamental biochemical functions of phototrophic cells. Significance statement By applying novel live microscopy monitoring using fluorescent protein biosensors in plant cells, we reveal that dark-light transitions trigger profound re-orchestration of subcellular pH, ATP and NAD redox physiology not limited to chloroplasts but extending into the cytosol and the mitochondria. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, 386512654, 507704013, 289357231 China Scholarship Council, 202006910019 [1]: #ref-2

In eukaryotic cells, protein supply to organelles varies depending on the stage of development and, in particular, exposure to environmental challenges. Adequate protein supply in terms of quality and quantity relies on sophisticated retrograde signalling systems that enable appropriate responses to the respective stress situations. Among many other retrograde signals, reactive oxygen species, which are generated during the initial stress response, are thought to be involved in transduction of redox-related signals that may also involve multiple redox pairs such as NAD(P)H/NAD(P)+ and redox-active metabolites such as glutathione. Deciphering such signals requires detailed knowledge of their amplitudes and temporal and spatial dynamics. Genetically encoded biosensors based on fluorescent proteins have been developed for a number of different redox-related physiological parameters and can be monitored in living cells, tissues, and even whole plants using a variety of instruments adapted to the respective resolution requirements, thus opening gateways to retrograde signalling in plant cells. This review summarizes and critically evaluates current probes and devices used to monitor sensor fluorescence. It also outlines how biosensors can be used in combination with genetic and pharmacological approaches, to extract meaningful information and dissect the retrograde redox signalling systems in living plants.

Plant exposure to unfavourable environmental conditions causes stress and reduces productivity. A common consequence of stress responses, are increased levels of reactive oxygen species (ROS), which if not controlled, could eventually lead to oxidative stress, damaging lipids, proteins and DNA, and ultimately result in cell death. One of the multiple defense systems that plants employ to regulate intracellular ROS levels are glutathione transferases (GSTs). GSTs have multiple roles in mitigating oxidative stress, e.g., by detoxifying xenobiotics through conjugation with reduced glutathione (GSH) or by using GSH to reduce damaging lipid hydroperoxides. In plants, GSTs exist in particularly large families and frequently occur in tandem gene clusters. This promotes the idea of functional diversification among closely related GSTs. This review focuses on the roles of GSTs in mitigating oxidative stress in plants and mentions potential strategies for functional analysis of the importance of individual GSTs by dissecting their enzymatic activities.

Essential iron–sulfur cofactors are assembled by multiprotein machineries in most life forms. Now, in vitro reconstitution of the bacterial core machinery has provided snapshots of the early assembly steps and revealed that [2Fe–2S] clusters are formed from two [1Fe–1S] precursors.

Specific generation of reactive oxygen species (ROS) is important for signalling and defence in many organisms. In plants, different types of ROS serve useful biological functions in the extracellular space (apoplast), influencing polymer structures as well as signalling during immune responses. The current knowledge of apoplastic ROS dynamics is limited, as dynamic monitoring of extracellular redox processes in vivo remains difficult. We employed evolutionary distant land plant model species from bryophytes and flowering plants to test whether the genetically encoded redox biosensor roGFP2-Orp1 can be used to assess extracellular redox dynamics. Secreted roGFP2-Orp1 can provide information about local diffusion barriers and protein cysteinyl oxidation rate in the apoplast, after pre-reduction. Observed re-oxidation rates were slow - within the range of hours. Compared to Physcomitrium patens, re-oxidation in Arabidopsis thaliana was faster and increased after triggering an immune response. Comparing roGFP2-Orp1 signals in tip-growing P. patens protonema and Nicotiana tabacum pollen tubes, we consistently find no intracellular redox gradient, but a partially reduced extracellular sensor in pollen tubes. Our data indicate differences in extracellular oxidative processes between species and within a species, depending on cell type and immune signalling.

Over the last two decades, the development of fluorescent probes has transformed the way of measuring physiological parameters in intact cells, including in the field of redox biology. We developed a genetically encoded biosensor called CyReB to monitor intracellular cysteine in real time. This biosensor exploits the ability of a particular bacterial cysteine desulfurase to promote the oxidation of reduction-oxidation-sensitive green fluorescent protein 2 in the presence of cysteine. The specificity, sensitivity, and the oxidation-reduction dynamics of CyReB were first investigated in vitro before its in vivo functionality was confirmed by expressing CyReB in Escherichia coli and Saccharomyces cerevisiae cells. Expressing CyReB or an inactive version in wild-type and various mutant strains of Escherichia coli showed that this sensor could be used to monitor intracellular cysteine dynamics, particularly in the context of the cysteine-cystine shuttle system. This work demonstrates how using this cysteine biosensor should provide new insights into the metabolism of cysteine and cysteine-related pathways in various model organisms.

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens.While we find that PpGRXC5 has high activities in GSH-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 as substrates in vitro, Δgrxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH (Δgr1). Using stroma-targeted roGFP2, we show increased protein Cys steady state oxidation and decreased reduction rates after oxidative challenge in Δgrxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from EGSH. Compared to wildtype, protein Cys disulfide formation rates and S-glutathionylation levels after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation.Our observations suggest specific roles for GRXC5 in the efficient transfer of electrons from GSH to target protein Cys as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function in efficient catalysis of protein dithiol/disulfide equilibria upon redox steady state alterations affecting stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5.

Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to F- cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron-electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the MgII-bound apo, and the F--bound holo form. 19F ENDOR (electron-nuclear double resonance) was used to investigate the active site structure of the F--bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing longer distances, revealed varying conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub-angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques.

Posttranslational modifications (PTMs) can modulate the activity, localization and interactions of proteins and (re)define their biological function. Understanding how changing environments can alter cellular processes thus requires detailed knowledge about the dynamics of PTMs in time and space. A PTM that gained increasing attention in the last decades is protein persulfidation, where a cysteine thiol (-SH) is covalently bound to sulfane sulfur to form a persulfide (-SSH). The precise cellular mechanisms underlying the presumed persulfide signaling in plants are, however, only beginning to emerge. In the mitochondrial matrix, strict regulation of persulfidation and H2S homeostasis is of prime importance for maintaining mitochondrial bioenergetic processes because H2S is a highly potent poison for cytochrome c oxidase. This review summarizes the current knowledge about protein persulfidation and corresponding processes in mitochondria of the model plant Arabidopsis. These processes will be compared to the respective processes in non-plant models to underpin similarities or highlight apparent differences. We provide an overview of mitochondrial pathways that contribute to H2S and protein persulfide generation and mechanisms for H2S fixation and de-persulfidation. Based on current proteomic data, we compile a plant mitochondrial persulfidome and discuss how persulfidation may regulate protein function.

Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness. Barley plants stably expressing Grx1-roGFP2 show high robustness of cytosolic glutathione redox potential under combined salt and osmotic stress, but respond with oxidation after infiltration with the Botrytis cinerea phytotoxic secretome.

In contrast to its close homolog PLANT UNCOUPLING MITOCHONDRIAL PROTEIN 1 (UCP1), which is an abundant carrier protein in the mitochondria, UCP2 localizes to the Golgi.

SUMMARYClass 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.

The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.

After an initial evolution in a reducing environment, life got successively challenged by reactive oxygen species (ROS), especially during the great oxidation event (GOE) that followed the development of photosynthesis. Therefore, ROS are deeply intertwined into the physiological, morphological and transcriptional responses of most present-day organisms. Copper-zinc superoxide dismutases (CuZnSODs) evolved during the GOE and are present in charophytes and extant land plants, but nearly absent from chlorophytes. The chemical inhibitor of CuZnSOD, lung cancer screen 1 (LCS-1), could greatly facilitate the study of SODs in diverse plants. Here, we determined the impact of chemical inhibition of plant CuZnSOD activity, on plant growth, transcription and metabolism. We followed a comparative approach by using different plant species, including Marchantia Polymorpha and Physcomitrium patens, representing bryophytes, the sister lineage to vascular plants, and Arabidopsis thaliana. We show that LCS-1 causes oxidative stress in plants and that the inhibition of CuZnSODs provoked a similar core response that mainly impacted glutathione homoeostasis in all plant species analysed. That said, Physcomitrium and Arabidopsis, which contain multiple CuZnSOD isoforms showed a more complex and exacerbated response. In addition, an untargeted metabolomics approach revealed a specific metabolic signature for each plant species. Our comparative analysis exposes a conserved core response at the physiological and transcriptional level towards LCS-1, while the metabolic response largely varies. These differences correlate with the number and localization of the CuZnSOD isoforms present in each species.

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.

Chlorophyll degradation causes the release of phytol, which is converted into phytyl diphosphate (phytyl-PP) by phytol kinase (VITAMIN E PATHWAY GENE5 [VTE5]) and phytyl phosphate (phytyl-P) kinase (VTE6). The kinase pathway is important for tocopherol synthesis, as the Arabidopsis (Arabidopsis thaliana) vte5 mutant contains reduced levels of tocopherol. Arabidopsis harbors one paralog of VTE5, farnesol kinase (FOLK) involved in farnesol phosphorylation. Here, we demonstrate that VTE5 and FOLK harbor kinase activities for phytol, geranylgeraniol, and farnesol with different specificities. While the tocopherol content of the folk mutant is unchanged, vte5-2 folk plants completely lack tocopherol. Tocopherol deficiency in vte5-2 plants can be complemented by overexpression of FOLK, indicating that FOLK is an authentic gene of tocopherol synthesis. The vte5-2 folk plants contain only ∼40% of wild-type amounts of phylloquinone, demonstrating that VTE5 and FOLK both contribute in part to phylloquinone synthesis. Tocotrienol and menaquinone-4 were produced in vte5-2 folk plants after supplementation with homogentisate or 1,4-dihydroxy-2-naphthoic acid, respectively, indicating that their synthesis is independent of the VTE5/FOLK pathway. These results show that phytyl moieties for tocopherol synthesis are completely but, for phylloquinone production, only partially derived from geranylgeranyl-chlorophyll and phytol phosphorylation by VTE5 and FOLK.

The escalating impacts of climate change on the movement and immobility of people, coupled with false but influential narratives of mobility, highlight an urgent need for nuanced and synthetic research around climate mobility. Synthesis of evidence and gaps across the Intergovernmental Panel on Climate Change (IPCC) Sixth Assessment Report highlight a need to clarify the understanding of what conditions make human mobility an effective adaptation option and its nuanced outcomes, including simultaneous losses, damages, and benefits. Priorities include integration of adaptation and development planning; involuntary immobility and vulnerability; gender; data for cities; risk from responses and maladaptation; public understanding of climate risk; transboundary, compound, and cascading risks; nature-based approaches; and planned retreat, relocation, and heritage. Cutting across these priorities, research modalities need to better position climate mobility as type of mobility, as process, and as praxis. Policies and practices need to reflect the diverse needs, priorities, and experiences of climate mobility, emphasizing capability, choice, and freedom of movement.