Alternative electron sinks in chloroplasts and mitochondria of halophytes as a safety valve for controlling ROS production during salinity.

Electron flow through the electron transport chain (ETC) is essential for oxidative phosphorylation in mitochondria and photosynthesis in chloroplasts. Electron fluxes depend on environmental parameters, e.g., ionic and osmotic conditions and endogenous factors, and this may cause severe imbalances. Plants have evolved alternative sinks to balance the reductive load on the electron transport chains in order to avoid overreduction, generation of reactive oxygen species (ROS), and to cope with environmental stresses. These sinks act primarily as valves for electron drainage and secondarily as regulators of tolerance-related metabolism, utilizing the excess reductive energy. High salinity is an environmental stressor that stimulates the generation of ROS and oxidative stress, which affects growth and development by disrupting the redox homeostasis of plants. While glycophytic plants are sensitive to high salinity, halophytic plants tolerate, grow, and reproduce at high salinity. Various studies have examined the ETC systems of glycophytic plants, however, information about the state and regulation of ETCs in halophytes under non-saline and saline conditions is scarce. This review focuses on alternative electron sinks in chloroplasts and mitochondria of halophytic plants. In cases where information on halophytes is lacking, we examined the available knowledge on the relationship between alternative sinks and gradual salinity resilience of glycophytes. To this end, transcriptional responses of involved components of photosynthetic and respiratory ETCs were compared between the glycophyte Arabidopsis thaliana and the halophyte Schrenkiella parvula, and the time-courses of these transcripts were examined in A. thaliana. The observed regulatory patterns are discussed in the context of reactive molecular species formation in halophytes and glycophytes.

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.

Development of an efficient and scalable bioprocess for the plant hormone 12-OPDA: Overcoming the hurdles of nature's biosynthesis.

Besides its native biological function as a plant hormone, cis-(+)-12-oxo-phytodienoic acid (12-OPDA) serves as a metabolite for the cellular formation of (-)-jasmonic acid and has also been shown to have an influence on mammalian cells. In order to make this biologically active, but at the same time very expensive natural product 12-OPDA broadly accessible for further biological and medicinal research, we developed an efficient bioprocess based on the utilization of a tailor-made whole-cell catalyst by following the principles of its biosynthesis in nature. After process optimization, the three-step one-pot synthesis of 12-OPDA starting from readily accessible α-linolenic acid could be conducted at appropriate technically relevant substrate loadings in the range of 5-20 g L-1. The desired 12-OPDA was obtained with an excellent conversion efficiency, and by means of the developed, efficient downstream-processing, this emulsifying as well as stereochemically labile biosynthetic metabolite 12-OPDA was then obtained with very high chemical purity (>99%) and enantio- and diastereomeric excess (>99% ee, 96% de) as well as negligible side-product formation (<1%). With respect to future technical applications, we also demonstrated the scalability of the production of the whole cell-biocatalyst in a high cell-density fermentation process.

Strategies of Molecular Signal Integration for Optimized Plant Acclimation to Stress Combinations.

Plant growth and survival in their natural environment require versatile mitigation of diverse threats. The task is especially challenging due to the largely unpredictable interaction of countless abiotic and biotic factors. To resist an unfavorable environment, plants have evolved diverse sensing, signaling, and adaptive molecular mechanisms. Recent stress studies have identified molecular elements like secondary messengers (ROS, Ca2+, etc.), hormones (ABA, JA, etc.), and signaling proteins (SnRK, MAPK, etc.). However, major gaps remain in understanding the interaction between these pathways, and in particular under conditions of stress combinations. Here, we highlight the challenge of defining "stress" in such complex natural scenarios. Therefore, defining stress hallmarks for different combinations is crucial. We discuss three examples of robust and dynamic plant acclimation systems, outlining specific plant responses to complex stress overlaps. (a) The high plasticity of root system architecture is a decisive feature in sustainable crop development in times of global climate change. (b) Similarly, broad sensory abilities and apparent control of cellular metabolism under adverse conditions through retrograde signaling make chloroplasts an ideal hub. Functional specificity of the chloroplast-associated molecular patterns (ChAMPs) under combined stresses needs further focus. (c) The molecular integration of several hormonal signaling pathways, which bring together all cellular information to initiate the adaptive changes, needs resolving.

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.

Reprogramming the translatome during daily light transitions as affected by cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1/C2.

Dark-light and light-dark transitions during the day are switching points of leaf metabolism that strongly affect the regulatory state of the cells, and this change is hypothesized to affect the translatome. The cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1 and GAPC2 function in glycolysis, and carbohydrate and energy metabolism, but GAPC1/C2 also shows moonlighting functions in gene expression and post-transcriptional regulation. In this study we examined the rapid reprogramming of the translatome that occurs within 10 min at the end of the night and the end of the day in wild-type (WT) Arabidopsis and a gapc1/c2 double-knockdown mutant. Metabolite profiling compared to the WT showed that gapc1/c2 knockdown led to increases in a set of metabolites at the start of day, particularly intermediates of the citric acid cycle and linked pathways. Differences in metabolite changes were also detected at the end of the day. Only small sets of transcripts changed in the total RNA pool; however, RNA-sequencing revealed major alterations in polysome-associated transcripts at the light-transition points. The most pronounced difference between the WT and gapc1/c2 was seen in the reorganization of the translatome at the start of the night. Our results are in line with the proposed hypothesis that GAPC1/C2 play a role in the control of the translatome during light/dark transitions.

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.

Oxylipins and Reactive Carbonyls as Regulators of the Plant Redox and Reactive Oxygen Species Network under Stress.

Reactive oxygen species (ROS), and in particular H2O2, serve as essential second messengers at low concentrations. However, excessive ROS accumulation leads to severe and irreversible cell damage. Hence, control of ROS levels is needed, especially under non-optimal growth conditions caused by abiotic or biotic stresses, which at least initially stimulate ROS synthesis. A complex network of thiol-sensitive proteins is instrumental in realizing tight ROS control; this is called the redox regulatory network. It consists of sensors, input elements, transmitters, and targets. Recent evidence revealed that the interplay of the redox network and oxylipins-molecules derived from oxygenation of polyunsaturated fatty acids, especially under high ROS levels-plays a decisive role in coupling ROS generation and subsequent stress defense signaling pathways in plants. This review aims to provide a broad overview of the current knowledge on the interaction of distinct oxylipins generated enzymatically (12-OPDA, 4-HNE, phytoprostanes) or non-enzymatically (MDA, acrolein) and components of the redox network. Further, recent findings on the contribution of oxylipins to environmental acclimatization will be discussed using flooding, herbivory, and establishment of thermotolerance as prime examples of relevant biotic and abiotic stresses.

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.

The mutual interaction of glycolytic enzymes and RNA in post-transcriptional regulation.

About three decades ago, researchers suggested that metabolic enzymes participate in cellular processes that are unrelated to their catalytic activity, and the term "moonlighting functions" was proposed. Recently developed advanced technologies in the field of RNA interactome capture now unveil the unexpected RNA binding activity of many metabolic enzymes, as exemplified here for the enzymes of glycolysis. Although for most of these proteins a precise binding mechanism, binding conditions, and physiological relevance of the binding events still await in-depth clarification, several well explored examples demonstrate that metabolic enzymes hold crucial functions in post-transcriptional regulation of protein synthesis. This widely conserved RNA-binding function of glycolytic enzymes plays major roles in controlling cell activities. The best explored examples are glyceraldehyde 3-phosphate dehydrogenase, enolase, phosphoglycerate kinase, and pyruvate kinase. This review summarizes current knowledge about the RNA-binding activity of the ten core enzymes of glycolysis in plant, yeast, and animal cells, its regulation and physiological relevance. Apparently, a tight bidirectional regulation connects core metabolism and RNA biology, forcing us to rethink long established functional singularities.

Mitochondrial Peroxiredoxin-IIF (PRXIIF) Activity and Function during Seed Aging.

Mitochondria play a major role in energy metabolism, particularly in cell respiration, cellular metabolism, and signal transduction, and are also involved in other processes, such as cell signaling, cell cycle control, cell growth, differentiation and apoptosis. Programmed cell death is associated with the production of reactive oxygen species (ROS) and a concomitant decrease in antioxidant capacity, which, in turn, determines the aging of living organisms and organs and thus also seeds. During the aging process, cell redox homeostasis is disrupted, and these changes decrease the viability of stored seeds. Mitochondrial peroxiredoxin-IIF (PRXIIF), a thiol peroxidase, has a significant role in protecting the cell and sensing oxidative stress that occurs during the disturbance of redox homeostasis. Thioredoxins (TRXs), which function as redox transmitters and switch protein function in mitochondria, can regulate respiratory metabolism. TRXs serve as electron donors to PRXIIF, as shown in Arabidopsis. In contrast, sulfiredoxin (SRX) can regenerate mitochondrial PRXIIF once hyperoxidized to sulfinic acid. To protect against oxidative stress, another type of thiol peroxidases, glutathione peroxidase-like protein (GPXL), is important and receives electrons from the TRX system. They remove peroxides produced in the mitochondrial matrix. However, the TRX/PRX and TRX/GPXL systems are not well understood in mitochondria. Knowledge of both systems is important because these systems play an important role in stress sensing, response and acclimation, including redox imbalance and generation of ROS and reactive nitrogen species (RNS). The TRX/PRX and TRX/GPXL systems are important for maintaining cellular ROS homeostasis and maintaining redox homeostasis under stress conditions. This minireview focuses on the functions of PRXIIF discovered in plant cells approximately 20 years ago and addresses the question of how PRXIIF affects seed viability maintenance and aging. Increasing evidence suggests that the mitochondrial PRXIIF plays a major role in metabolic processes in seeds, which was not previously known.

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.