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.

Inactivation of Three Stacked Genes of Cytosolic Peroxiredoxins by Genome Editing.

A set of peroxidases detoxifies H2O2 and mediates H2O2-dependent signal propagation. The peroxidases include peroxiredoxins, glutathione peroxidases, ascorbate peroxidases, and catalases. This at least partial redundancy impedes addressing individual proteins in living plant cells so that the protein functions are often studied by biochemical assays in vitro. In vivo analysis frequently relies on transgenic insertion lines resulting in the knockdown or knockout of the protein of interest. However, many proteins have multiple isoforms in close genomic arrangement so that even crossing of transgenic lines does not allow for a knockdown of all isoforms. The genes encoding for the three cytosolic peroxiredoxins PRXIIB, C, and D in Arabidopsis thaliana are located in close vicinity on chromosome 1 so that crossing over between the genes most rarely occurs and successful crossing of the plants appears impossible. Genome editing instead allows targeting of multiple isoforms and knocks out several genes at once. This chapter describes how to inactivate the three cytosolic peroxiredoxins by CRISPR/Cas9 in A. thaliana.

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.

The centrality of redox regulation and reactive oxygen species sensing in abiotic and biotic stress acclimatization.

During land plant evolution, the number of genes encoding for components of the thiol redox regulatory network and the generator systems of reactive oxygen species (ROS) expanded, tentatively indicating a role in tailored environmental acclimatization. This hypothesis has been validated experimentally and theoretically during the last decades. Recent developments of dynamic roGFP-based in vivo sensors for H2O2 and the redox potential of the glutathione pool paved the way for dissecting the kinetics changes in these decisive parameters in response to environmental stressors. The versatile cellular redox sensory and response regulatory system monitors alterations in redox metabolism and controls the activity of redox target proteins, and thereby affects most, if not all, cellular processes ranging from transcription to translation and metabolism. This review exemplarily describes the role of the redox- and ROS-dependent regulatory network in realising the proper response to diverse environmental stresses. The selected case studies concern different environmental challenges, namely excess excitation energy, the heavy metal cadmium and the metalloid arsenic, nitrogen, or phosphate shortage as examples for nutrient deficiency, wounding, and nematode infestation. Each challenge affects the redox regulatory and ROS network, but the present state of knowledge also pinpoints to pressing open questions concerning the translation of redox regulation to environmental acclimatization.

A general concept of quantitative abiotic stress sensing.

Plants often encounter stress in their environment. For appropriate responses to particular stressors, cells rely on sensory mechanisms that detect emerging stress. Considering sensor and signal amplification characteristics, a single sensor system hardly covers the entire stress range encountered by plants (e.g., salinity, drought, temperature stress). A dual system comprising stress-specific sensors and a general quantitative stress sensory system is proposed to enable the plant to optimize its response. The quantitative stress sensory system exploits the redox and reactive oxygen species (ROS) network by altering the oxidation and reduction rates of individual redox-active molecules under stress impact. The proposed mechanism of quantitative stress sensing also fits the requirement of dealing with multifactorial stress conditions.

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.

Regeneration of cytosolic thiol peroxidases.

Three soluble type two peroxiredoxins (PRXIIB, C, D) and two glutathione peroxidase-like enzymes (GPXL2, 8) reside in the cytosol of Arabidopsis thaliana cells and function both as thiol-dependent antioxidants and redox sensors. Their primary substrate is H2 O2 , but they also accept other peroxides with a distinct preference between PRXII and GPXL. Less known is their regeneration specificity in the light of the large set of thiol reductases, namely eight annotated thioredoxin h isoforms (TRXh1-5, 7-9), a few TRX-like proteins, including CxxS1 (formerly TRXh6) and several glutaredoxins (GRX) associated with the cytosol. This study addressed this open question by in vitro enzyme tests using recombinant protein. GPXL2 and 8 exclusively accepted electrons from the TRX system, namely TRXh1-5 and TDX, while PRXIIB/C/D were efficiently regenerated with GRXC1 and C2 but not the TRX-like protein Picot1. They showed significant but low activity (<3% of GRXC2) with TRXh1-5 and TDX. A similar reduction efficiency with TRX was seen in the insulin assay, only TDX was less active. Finally, the reduction of oxidized cytosolic malate dehydrogenase 1, as measured by regained activity, showed an extremely broad ability to accept electrons from different TRXs and GRXs. The results demonstrate redundancy and specificity in the redox regulatory network of the cytosol.

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.

Remodeling of Root Growth Under Combined Arsenic and Hypoxia Stress Is Linked to Nutrient Deprivation.

Root architecture responds to environmental stress. Stress-induced metabolic and nutritional changes affect the endogenous root development program. Transcriptional and translational changes realize the switch between stem cell proliferation and cell differentiation, lateral root or root hair formation and root functionality for stress acclimation. The current work explores the effects of stress combination of arsenic toxicity (As) and hypoxia (Hpx) on root development in Arabidopsis thaliana. As revealed previously, combined As and Hpx treatment leads to severe nutritional disorder evident from deregulation of root transcriptome and plant mineral contents. Both As and Hpx were identified to pose stress-specific constraints on root development that lead to unique root growth phenotype under their combination. Besides inhibition of root apical meristem (RAM) activity under all stresses, As induced lateral root growth while root hair density and lengths were strongly increased by Hpx and HpxAs-treatments. A dual stimulation of phosphate (Pi)-starvation response was observed for HpxAs-treated plant roots; however, the response under HpxAs aligned more with Hpx than As. Transcriptional evidence along with biochemical data suggests involvement of PHOSPHATE STARVATION RESPONSE 1; PHR1-dependent systemic signaling. Pi metabolism-related transcripts in close association with cellular iron homeostasis modulate root development under HpxAs. Early redox potential changes in meristematic cells, differential ROS accumulation in root hair zone cell layers and strong deregulation of NADPH oxidases, NADPH-dependent oxidoreductases and peroxidases signify a role of redox and ROS signaling in root architecture remodeling under HpxAs. Differential aquaporin expression suggests transmembrane ROS transport to regulate root hair induction and growth. Reorganization of energy metabolism through NO-dependent alternate oxidase, lactate fermentation, and phosphofructokinase seems crucial under HpxAs. TOR and SnRK-signaling network components were potentially involved in control of sustainable utilization of available energy reserves for root hair growth under combined stress as well as recovery on reaeration. Findings are discussed in context of combined stress-induced signaling in regulation of root development in contrast to As and Hpx alone.

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.

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.

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.