Thiol Redox Proteomics for Identifying Redox-Sensitive Cysteine Residues Within the Protein of Interest During Stress.

Redox modulation is a common posttranslational modification to regulate protein activity. The targets of oxidizing agents are cysteine residues (Cys), which have to be exposed at the surface of the proteins and are characterized by an environment that favors redox modulation. This includes their protonation state and the neighboring amino acids. The Cys redox state can be assessed experimentally by redox titrations to determine the midpoint redox potential in the protein. Exposed cysteine residues and putative intramolecular disulfide bonds can be predicted by alignments with structural data using dedicated software tools and information on conserved cysteine residues. Labeling with light and heavy reagents, such as N-ethylmaleimide (NEM), followed by mass spectrometric analysis, allows for the experimental determination of redox-responsive cysteine residues. This type of thiol redox proteomics is a powerful approach to assessing the redox state of the cell, e.g., in dependence on environmental conditions and, in particular, under abiotic stress.

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution.

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.

Chemoselective umpolung of thiols to episulfoniums for cysteine bioconjugation.

Cysteine conjugation is an important tool in protein research and relies on fast, mild and chemoselective reactions. Cysteinyl thiols can either be modified with prefunctionalized electrophiles, or converted into electrophiles themselves for functionalization with selected nucleophiles in an independent step. Here we report a bioconjugation strategy that uses a vinyl thianthrenium salt to transform cysteine into a highly reactive electrophilic episulfonium intermediate in situ, to enable conjugation with a diverse set of bioorthogonal nucleophiles in a single step. The reactivity profile can connect several nucleophiles to biomolecules through a short and stable ethylene linker, ideal for introduction of infrared labels, post-translational modifications or NMR probes. In the absence of reactive exogenous nucleophiles, nucleophilic amino acids can react with the episulfonium intermediate for native peptide stapling and protein-protein ligation. Ready synthetic access to isotopologues of vinyl thianthrenium salts enables applications in quantitative proteomics. Such diverse applications demonstrate the utility of vinyl-thianthrenium-based bioconjugation as a fast, selective and broadly applicable tool for chemical biology.

Plant thiol peroxidases as redox sensors and signal transducers in abiotic stress acclimation.

The temporal and spatial patterns of reactive oxygen species (ROS) in cells and tissues decisively determine the plant acclimation response to diverse abiotic and biotic stresses. Recent progress in developing dynamic cell imaging probes provides kinetic information on changes in parameters like H2O2, glutathione (GSH/GSSG) and NAD(P)H/NAD(P)+, that play a crucial role in tuning the cellular redox state. Central to redox-based regulation is the thiol-redox regulatory network of the cell that integrates reductive information from metabolism and oxidative ROS signals. Sensitive proteomics allow for monitoring changes in redox-related posttranslational modifications. Thiol peroxidases act as sensitive peroxide and redox sensors and play a central role in this signal transduction process. Peroxiredoxins (PRX) and glutathione peroxidases (GPX) are the two main thiol peroxidases and their function in ROS sensing and redox signaling in plants is emerging at present and summarized in this review. Depending on their redox state, PRXs and GPXs act as redox-dependent binding partners, direct oxidants of target proteins and oxidants of thiol redox transmitters that in turn oxidize target proteins. With their versatile functions, the multiple isoforms of plant thiol peroxidases play a central role in plant stress acclimation, e.g. to high light or osmotic stress, but also in ROS-mediated immunity and development.

Differential sensitivity of metabolic pathways in sugar beet roots to combined salt, heat, and light stress.

Plants in nature commonly encounter combined stress scenarios. The response to combined stressors is often unpredictable from the response to single stresses. To address stress interference in roots, we applied salinity, heat, and high light to hydroponically grown sugar beet. Two main patterns of metabolomic acclimation were apparent. High salt of 300 mM NaCl considerably lowered metabolite amounts, for example, those of most amino acids, γ-amino butyric acid (GABA), and glucose. Very few metabolites revealed the opposite trend with increased contents at high salts, mostly organic acids such as citric acid and isocitric acid, but also tryptophan, tyrosine, and the compatible solute proline. High temperature (31°C vs. 21°C) also frequently lowered root metabolite pools. The individual effects of salinity and heat were superimposed under combined stress. Under high light and high salt conditions, there was a significant decline in root chloride, mannitol, ribulose 5-P, cysteine, and l-aspartate contents. The results reveal the complex interaction pattern of environmental parameters and urge researchers to elaborate in much more detail and width on combinatorial stress effects to bridge work under controlled growth conditions to growth in nature, and also to better understand acclimation to the consequences of climate change.

OPDAylation of Thiols of the Redox Regulatory Network In Vitro.

cis-(+)-12-Oxophytodienoic acid (OPDA) is a reactive oxylipin produced by catalytic oxygenation of polyunsaturated α-linolenic acid (18:3 (ω - 3)) in the chloroplast. Apart from its function as precursor for jasmonic acid synthesis, OPDA serves as a signaling molecule and regulator on its own, namely by tuning enzyme activities and altering expression of OPDA-responsive genes. A possible reaction mechanism is the covalent binding of OPDA to thiols via the addition to the C=C double bond of its α,ß-unsaturated carbonyl group in the cyclopentenone ring. The reactivity allows for covalent modification of accessible cysteinyl thiols in proteins. This work investigated the reaction of OPDA with selected chloroplast and cytosolic thioredoxins (TRX) and glutaredoxins (GRX) of Arabidopsis thaliana. OPDA reacted with TRX and GRX as detected by decreased m-PEG maleimide binding, consumption of OPDA, reduced ability for insulin reduction and inability to activate glyceraldehyde-3-phosphate dehydrogenase and regenerate glutathione peroxidase (GPXL8), and with lower efficiency, peroxiredoxin IIB (PRXIIB). OPDAylation of certain protein thiols occurs quickly and efficiently in vitro and is a potent post-translational modification in a stressful environment.

Scrutinizing the Impact of Alternating Electromagnetic Fields on Molecular Features of the Model Plant Arabidopsis thaliana.

Natural and anthropogenic electromagnetic fields (EMFs) are ubiquitous in the environment and interfere with all biological organisms including plants. Particularly the quality and quantity of alternating EMFs from anthropogenic sources are increasing due to the implementation of novel technologies. There is a significant interest in exploring the impact of EMFs (similar to those emitted from battery chargers of electric cars) on plants. The model plant Arabidopsis thaliana was exposed to a composite alternating EMF program for 48 h and scrutinized for molecular alterations using photosynthetic performance, metabolite profiling, and RNA sequencing followed by qRT-PCR validation. Clear differences in the photosynthetic parameters between the treated and control plants indicated either lower nonphotochemical quenching or higher reduction of the plastoquinone pool or both. Transcriptome analysis by RNA sequencing revealed alterations in transcript amounts upon EMF exposure; however, the gene ontology groups of, e.g., chloroplast stroma, thylakoids, and envelope were underrepresented. Quantitative real-time PCR validated deregulation of some selected transcripts. More profound were the readjustments in metabolite pool sizes with variations in photosynthetic and central energy metabolism. These findings together with the invariable phenotype indicate efficient adjustment of the physiological state of the EMF-treated plants, suggesting testing for more challenging growth conditions in future experiments.

Single molecule mass photometry reveals the dynamic oligomerization of human and plant peroxiredoxins.

Protein oligomerization is central to biological function and regulation, yet its experimental quantification and measurement of dynamic transitions in solution remain challenging. Here, we show that single molecule mass photometry quantifies affinity and polydispersity of heterogeneous protein complexes in solution. We demonstrate these capabilities by studying the functionally relevant oligomeric equilibria of 2-cysteine peroxiredoxins (2CPs). Comparison of the polydispersity of plant and human 2CPs as a function of concentration and redox state revealed features conserved among all 2CPs. In addition, we also find species-specific differences in oligomeric transitions, the occurrence of intermediates and the formation of high molecular weight complexes, which are associated with chaperone activity or act as a storage pool for more efficient dimers outlining the functional differentiation of human 2CPs. Our results point to a diversified functionality of oligomerization for 2CPs and illustrate the power of mass photometry for characterizing heterogeneous oligomeric protein distributions in near native conditions.

The In Vitro Interaction of 12-Oxophytodienoic Acid and Related Conjugated Carbonyl Compounds with Thiol Antioxidants.

α,ß-unsaturated carbonyls interfere with numerous plant physiological processes. One mechanism of action is their reactivity toward thiols of metabolites like cysteine and glutathione (GSH). This work aimed at better understanding these interactions. Both 12-oxophytodienoic acid (12-OPDA) and abscisic acid (ABA) conjugated with cysteine. It was found that the reactivity of α,ß-unsaturated carbonyls with GSH followed the sequence trans-2-hexenal < 12-OPDA ≈ 12-OPDA-ethylester < 2-cyclopentenone << methyl vinylketone (MVK). Interestingly, GSH, but not ascorbate (vitamin C), supplementation ameliorated the phytotoxic potential of MVK. In addition, 12-OPDA and 12-OPDA-related conjugated carbonyl compounds interacted with proteins, e.g., with members of the thioredoxin (TRX)-fold family. 12-OPDA modified two cysteinyl residues of chloroplast TRX-f1. The OPDAylated TRX-f1 lost its activity to activate the Calvin-Benson-cycle enzyme fructose-1,6-bisphosphatase (FBPase). Finally, we show that 12-OPDA interacts with cyclophilin 20-3 (Cyp20-3) non-covalently and affects its peptidyl-prolyl-cis/trans isomerase activity. The results demonstrate the high potential of 12-OPDA as a diverse interactor and cellular regulator and suggest that OPDAylation may occur in plant cells and should be investigated as novel regulatory mechanism.

Shifting paradigms and novel players in Cys-based redox regulation and ROS signaling in plants - and where to go next.

Cys-based redox regulation was long regarded a major adjustment mechanism of photosynthesis and metabolism in plants, but in the recent years, its scope has broadened to most fundamental processes of plant life. Drivers of the recent surge in new insights into plant redox regulation have been the availability of the genome-scale information combined with technological advances such as quantitative redox proteomics and in vivo biosensing. Several unexpected findings have started to shift paradigms of redox regulation. Here, we elaborate on a selection of recent advancements, and pinpoint emerging areas and questions of redox biology in plants. We highlight the significance of (1) proactive H2O2 generation, (2) the chloroplast as a unique redox site, (3) specificity in thioredoxin complexity, (4) how to oxidize redox switches, (5) governance principles of the redox network, (6) glutathione peroxidase-like proteins, (7) ferroptosis, (8) oxidative protein folding in the ER for phytohormonal regulation, (9) the apoplast as an unchartered redox frontier, (10) redox regulation of respiration, (11) redox transitions in seed germination and (12) the mitochondria as potential new players in reductive stress safeguarding. Our emerging understanding in plants may serve as a blueprint to scrutinize principles of reactive oxygen and Cys-based redox regulation across organisms.

ConCysFind: a pipeline tool to predict conserved amino acids of protein sequences across the plant kingdom.

BACKGROUND: Post-translational modifications (PTM) of amino acid (AA) side chains in peptides control protein structure and functionality. PTMs depend on the specific AA characteristics. The reactivity of cysteine thiol-based PTMs are unique among all proteinaceous AA. This pipeline aims to ease the identification of conserved AA of polypeptides or protein families based on the phylogenetic occurrence in the plant kingdom. The tool is customizable to include any species. The degree of AA conservation is taken as indicator for structural and functional significance, especially for PTM-based regulation. Further, this pipeline tool gives insight into the evolution of these potentially regulatory important peptides. RESULTS: The web-based or stand-alone pipeline tool Conserved Cysteine Finder (ConCysFind) was developed to identify conserved AA such as cysteine, tryptophan, serine, threonine, tyrosin and methionine. ConCysFind evaluates multiple alignments considering the proteome of 21 plant species. This exemplar study focused on Cys as evolutionarily conserved target for multiple redox PTM. Phylogenetic trees and tables with the compressed results of the scoring algorithm are generated for each Cys in the query polypeptide. Analysis of 33 translation elongation and release factors alongside of known redox proteins from Arabidopsis thaliana for conserved Cys residues confirmed the suitability of the tool for identifying conserved and functional PTM sites. Exemplarily, the redox sensitivity of cysteines in the eukaryotic release factor 1-1 (eRF1-1) was experimentally validated. CONCLUSION: ConCysFind is a valuable tool for prediction of new potential protein PTM targets in a broad spectrum of species, based on conserved AA throughout the plant kingdom. The identified targets were successfully verified through protein biochemical assays. The pipeline is universally applicable to other phylogenetic branches by customization of the database.

Three cytosolic NAD-malate dehydrogenase isoforms of Arabidopsis thaliana: on the crossroad between energy fluxes and redox signaling.

In yeast and animal cells, mitochondrial disturbances resulting from imbalances in the respiratory chain require malate dehydrogenase (MDH) activities for re-directing fluxes of reducing equivalents. In plants, in addition to mitochondria, plastids use malate valves to counterbalance and maintain redox-homeostasis. Arabidopsis expresses three cytosolic MDH isoforms, namely cyMDH1, cyMDH2, and cyMDH3, the latter possessing an N-terminal extension carrying a unique cysteine residue C2. In this study, redox-effects on activity and structure of all three cyMDH isoforms were analyzed in vitro. cyMDH1 and cyMDH2 were reversibly inactivated by diamide treatment, accompanied by dimerization via disulfide-bridge formation. In contrast, cyMDH3 forms dimers and higher oligomers upon oxidation, but its low specific activity is redox-independent. In the presence of glutathione, cyMDH1 and cyMDH2 are protected from dimerization and inactivation. In contrast, cyMDH3 still dimerizes but does not form oligomers any longer. From analyses of single and double cysteine mutants and structural modeling of cyMDH3, we conclude that the presence of C2 and C336 allows for multiple cross-links in the higher molecular mass complexes comprising disulfides within the dimer as well as between monomers of two different dimers. Furthermore, nuclear localization of cyMDH isoforms was significantly increased under oxidizing conditions in isolated Arabidopsis protoplasts, in particular of isoform cyMDH3. The unique cyMDH3 C2-C2-linked dimer is, therefore, a good candidate as a redox-sensor taking over moonlighting functions upon disturbances of energy metabolism, as shown previously for the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) where oxidative modification of the sensitive catalytic cysteine residues induces nuclear translocation.

Thiol Redox Regulation of Plant ß-Carbonic Anhydrase.

ß-carbonic anhydrases (ßCA) accelerate the equilibrium formation between CO2 and carbonate. Two plant ßCA isoforms are targeted to the chloroplast and represent abundant proteins in the range of >1% of chloroplast protein. While their function in gas exchange and photosynthesis is well-characterized in carbon concentrating mechanisms of cyanobacteria and plants with C4-photosynthesis, their function in plants with C3-photosynthesis is less clear. The presence of conserved and surface-exposed cysteinyl residues in the ßCA-structure urged to the question whether ßCA is subject to redox regulation. Activity measurements revealed reductive activation of ßCA1, whereas oxidized ßCA1 was inactive. Mutation of cysteinyl residues decreased ßCA1 activity, in particular C280S, C167S, C230S, and C257S. High concentrations of dithiothreitol or low amounts of reduced thioredoxins (TRXs) activated oxidized ßCA1. TRX-y1 and TRX-y2 most efficiently activated ßCA1, followed by TRX-f1 and f2 and NADPH-dependent TRX reductase C (NTRC). High light irradiation did not enhance ßCA activity in wildtype Arabidopsis, but surprisingly in ßca1 knockout plants, indicating light-dependent regulation. The results assign a role of ßCA within the thiol redox regulatory network of the chloroplast.

Redox Conformation-Specific Protein-Protein Interactions of the 2-Cysteine Peroxiredoxin in Arabidopsis.

2-Cysteine peroxiredoxins (2-CysPRX) are highly abundant thiol peroxidases in chloroplasts and play key roles in reactive oxygen species (ROS) defense and redox signaling. Peroxide-dependent oxidation of cysteines induces conformational changes that alter the ability for protein-protein interactions. For regeneration, 2-CysPRXs withdraw electrons from thioredoxins (TRXs) and participate in redox-dependent regulation by affecting the redox state of TRX-dependent targets, for example, in chloroplast metabolism. This work explores the redox conformation-specific 2-CysPRX interactome using an affinity-based pull down with recombinant variants arrested in specific quaternary conformations. This allowed us to address a critical and poorly explored aspect of the redox-regulatory network and showed that the interaction of TRXs, their interaction partners, and 2-CysPRX occur under contrasting redox conditions. A set of 178 chloroplast proteins were identified from leaf proteins and included proteins with functions in photosynthesis, carbohydrate, fatty acid and amino acid metabolism, and defense. These processes are known to be deregulated in plants devoid of 2-CysPRX. Selected enzymes like LIPOXYGENASE 2, CHLOROPLAST PROTEIN 12-1, CHORISMATE SYNTHASE, ß-CARBONIC ANHYDRASE, and FERREDOXIN-dependent GLUTAMATE SYNTHASE 1 were subjected to far Western, isothermal titration calorimetry, and enzyme assays for validation. The pull down fractions frequently contained TRXs as well as their target proteins, for example, FRUCTOSE-1,6-BISPHOSPHATASE and MALATE DEHYDROGENASE. The difference between TRX-dependent indirect interactions of TRX targets and 2-CysPRX and direct 2-CysPRX binding is hypothesized to be related to quaternary structure formation, where 2-CysPRX oligomers function as scaffold for complex formation, whereas TRX oxidase activity of 2-CysPRX controls the redox state of TRX-related enzyme activity.

Regulatory thiol oxidation in chloroplast metabolism, oxidative stress response and environmental signaling in plants.

The antagonism between thiol oxidation and reduction enables efficient control of protein function and is used as central mechanism in cellular regulation. The best-studied mechanism is the dithiol-disulfide transition in the Calvin Benson Cycle in photosynthesis, including mixed disulfide formation by glutathionylation. The adjustment of the proper thiol redox state is a fundamental property of all cellular compartments. The glutathione redox potential of the cytosol, stroma, matrix and nucleoplasm usually ranges between -300 and -320 mV. Thiol reduction proceeds by short electron transfer cascades consisting of redox input elements and redox transmitters such as thioredoxins. Thiol oxidation ultimately is linked to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Enhanced ROS production under stress shifts the redox network to more positive redox potentials. ROS do not react randomly but primarily with few specific redox sensors in the cell. The most commonly encountered reaction within the redox regulatory network however is the disulfide swapping. The thiol oxidation dynamics also involves transnitrosylation. This review compiles present knowledge on this network and its central role in sensing environmental cues with focus on chloroplast metabolism.

Computational simulation of the reactive oxygen species and redox network in the regulation of chloroplast metabolism.

Cells contain a thiol redox regulatory network to coordinate metabolic and developmental activities with exogenous and endogenous cues. This network controls the redox state and activity of many target proteins. Electrons are fed into the network from metabolism and reach the target proteins via redox transmitters such as thioredoxin (TRX) and NADPH-dependent thioredoxin reductases (NTR). Electrons are drained from the network by reactive oxygen species (ROS) through thiol peroxidases, e.g., peroxiredoxins (PRX). Mathematical modeling promises access to quantitative understanding of the network function and was implemented by using published kinetic parameters combined with fitting to known biochemical data. Two networks were assembled, namely the ferredoxin (FDX), FDX-dependent TRX reductase (FTR), TRX, fructose-1,6-bisphosphatase (FBPase) pathway with 2-cysteine PRX/ROS as oxidant, and separately the FDX, FDX-dependent NADP reductase (FNR), NADPH, NTRC-pathway for 2-CysPRX reduction. Combining both modules allowed drawing several important conclusions of network performance. The resting H2O2 concentration was estimated to be about 30 nM in the chloroplast stroma. The electron flow to metabolism exceeds that into thiol regulation of FBPase more than 7000-fold under physiological conditions. The electron flow from NTRC to 2-CysPRX is about 5.32-times more efficient than that from TRX-f1 to 2-CysPRX. Under severe stress (30 µM H2O2) the ratio of electron flow to the thiol network relative to metabolism sinks to 1:251 whereas the ratio of e- flow from NTRC to 2-CysPRX and TRX-f1 to 2-CysPRX rises up to 1:67. Thus, the simulation provides clues on experimentally inaccessible parameters and describes the functional state of the chloroplast thiol regulatory network.

Magnetic Tracking of Protein Synthesis in Microfluidic Environments-Challenges and Perspectives.

A novel technique to study protein synthesis is proposed that uses magnetic nanoparticles in combination with microfluidic devices to achieve new insights into translational regulation. Cellular protein synthesis is an energy-demanding process which is tightly controlled and is dependent on environmental and developmental requirements. Processivity and regulation of protein synthesis as part of the posttranslational nano-machinery has now moved back into the focus of cell biology, since it became apparent that multiple mechanisms are in place for fine-tuning of translation and conditional selection of transcripts. Recent methodological developments, such as ribosome foot printing, propel current research. Here we propose a strategy to open up a new field of labelling, separation, and analysis of specific polysomes using superparamagnetic particles following pharmacological arrest of translation during cell lysis and subsequent analysis. Translation occurs in polysomes, which are assemblies of specific transcripts, associated ribosomes, nascent polypeptides, and other factors. This supramolecular structure allows for unique approaches to selection of polysomes by targeting the specific transcript, ribosomes, or nascent polypeptides. Once labeled with functionalized superparamagnetic particles, such assemblies can be separated in microfluidic devices or magnetic ratchets and quantified. Insights into the dynamics of translation is obtained through quantifying large numbers of ribosomes along different locations of the polysome. Thus, an entire new concept for in vitro, ex vivo, and eventually single cell analysis will be realized and will allow for magnetic tracking of protein synthesis.

Thiol redox-regulation for efficient adjustment of sulfur metabolism in acclimation to abiotic stress.

Sulfur assimilation and sulfur metabolism are tightly controlled at the transcriptional, post-transcriptional, and post-translational levels in order to meet the demand for reduced sulfur in growth and metabolism. These regulatory mechanisms coordinate the cellular sulfhydryl supply with carbon and nitrogen assimilation in particular. Redox homeostasis is an important cellular parameter intimately connected to sulfur by means of multiple thiol modifications. Post-translational thiol modifications such as disulfide formation, sulfenylation, S-nitrosylation, persulfidation, and S-glutathionylation allow for versatile switching and adjustment of protein functions. This review focuses on redox-regulation of enzymes involved in the sulfur assimilation pathway, namely adenosine 5´-phosphosulfate reductase (APR), adenosine 5´-phosphosulfate kinase (APSK), and γ-glutamylcysteine ligase (GCL). The activity of these enzymes is adjusted at the transcriptional and post-translational level depending on physiological requirements and the state of the redox and reactive oxygen species network, which are tightly linked to abiotic stress conditions. Hormone-dependent fine-tuning contributes to regulation of sulfur assimilation. Thus, the link between oxylipin signalling and sulfur assimilation has been substantiated by identification of the so-called COPS module in the chloroplast with its components cyclophilin 20-3, O-acetylserine thiol lyase, 2-cysteine peroxiredoxin, and serine acetyl transferase. We now have a detailed understanding of how regulation enables the fine-tuning of sulfur assimilation under both normal and abiotic stress conditions.

Interference between arsenic-induced toxicity and hypoxia.

Plants often face combinatorial stresses in their natural environment. Here, arsenic (As) toxicity was combined with hypoxia (Hpx) in the roots of Arabidopsis thaliana as it often occurs in nature. Arsenic inhibited growth of both roots and leaves, whereas root growth almost entirely ceased in Hpx. Growth efficiently resumed, and Hpx marker transcripts decreased upon reaeration. Compromised recovery from HpxAs treatment following reaeration indicated some persistent effects of combined stresses despite lower As accumulation. Root glutathione redox potential turned more oxidized in Hpx and most strongly in HpxAs. The more oxidizing root cell redox potential and the lowered glutathione amounts may be conducive to the growth arrest of plants exposed to HpxAs. The stresses elicited changes in elemental and transcriptomic composition. Thus, calcium, magnesium, and phosphorous amounts decreased in rosettes, but the strongest decline was seen for potassium. The reorganized potassium-related transcriptome supports the conclusion that disturbed potassium homeostasis contributes to the growth phenotype. In a converse manner, photosynthesis-related parameters were hardly affected, whereas accumulated carbohydrates under all stresses and anthocyanins under Hpx exclude carbohydrate limitation. The study demonstrates the existence of both synergistic since mutually aggravating effects and antagonistic effects of single and combined stresses.