Persulfidation, a posttranslational modification of cysteines to persulfides, is the best characterized molecular mechanism of H2S signaling. This study is focused on new functions for thioredoxins (TRXs) in plants beyond those of thiol disulfide (S-S) exchange, including the regulation of protein persulfidation as it has been described in animal systems. To elucidate the impact of TRXo1 deficiency on the protein persulfidation pattern in plants of Arabidopsis thaliana L. wild type (WT) and two Attrxo1 T-DNA insertion mutants grown under non stress conditions, a quantitative proteomic approach was performed. The proteomic analysis revealed a higher number of proteins that were more persulfidated in the mutants compared to WT plants, suggesting a role for TRXo1 in protein depersulfidation. Interestingly, most of the differentially persulfidated proteins were located in the chloroplast, implying a coordination between chloroplast H2S-dependent persulfidation and mitochondrial TRXo1 depersulfidation. Among the differentially persulfidated proteins located in mitochondria, the antioxidant enzymes sAPX, DHAR1 and MDAR6 were selected for further studies. The effect of H2S-dependent persulfidation on their enzymatic activities and its reversibility by the NADPH/thioredoxin reductase (NTRB)/TRXo1 system was analyzed, as well as their persulfidation levels were quantified. Sulfide treatment brought about increases in the activity levels of the enzymes, that match with a raise on the persulfidation levels. Interestingly, both activations declining after treatment with the thioredoxin system, indicate the regulation of their persulfidation by TRXo1. These results point to a positive effect of persulfidation on the enzymatic activities and also to a new depersulfidase activity for TRXo1. All together these results give a new insight of the mechanism of elimination of -SSH groups in plants exerted by TRXo1, and the involvement of a redox regulation on the protein persulfidation.

Plant acclimation to changing environmental conditions involves the interaction of different signalling molecules, including reactive oxygen species and hormones. Redox regulation exerted by thioredoxin (TRX) and glutaredoxin (GRX), two oxidoreductases, is emerging as a specific point of control mediating signal transduction pathways associated with plant growth and stress response. Phytohormones are messengers that coordinate plant cell activities to regulate growth, defence, and productivity, although their cross-talk with components of the redox system is less known. The present review focuses on our current knowledge of the interplay that occurs between TRX and GRX systems and phytohormonal signalling pathways in connection with the control of plant development and stress responses. Here, we consider the regulation that phytohormones exert on TRX and GRX systems, as well as the involvement of these redox proteins in the control of phytohormone-mediated signalling pathways.

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.

An uncontrolled, natural episode of flooding with waters contaminated with As-rich pyrite (FeAsS) particles caused serious ecological damage leading to necrosis of plants growing in a fresh wet meadow located in an area characterized by unique geological structures rich in arsenopyrites. One of the few plant species capable of surviving this event was Salix aurita L., which grew in numbers in the analyzed area, but individual plants were affected differently by toxic flooding. No significant phenotypic changes (Group I), through partial leaf and/or stem necrosis (Group II) up to necrosis of the whole parental plant and root suckers (Group III), were observed for various willow clumps. These varied phenotypic responses of S. aurita to As-rich sediments were compared with the biochemical status of the foliage of willow trees, and with their rhizosphere physiological parameters. Our in situ study revealed that the biochemical status of leaves reflects the phenotypic damage incurred by adult willows growing in their natural environment and affected by the flooding. In leaves of willows with increasingly negative phenotypic changes (Groups I → II → III) as well as increasing levels of reactive oxygen species, malondialdehyde and decreased levels of glutathione and thiol groups were detected. Phytochelatins, commonly considered major As chelators, were not detected in S. aurita leaves. Despite a decrease in the size of leaves with the intensity of tree damage, all leaves expressed a normal level of leaf pigments. Phenotypic changes observed for particular willow clumps were only partly related to soil As levels. Moreover, As and S (but not Fe) foliar levels were related but did not correspond strictly with foliar biochemical features, or with soil As levels, soil pH or soil microbial activity, with the latter two drastically decreased in the rhizospheres of willows from Groups II and III.

Atriplex canescens is a representative halophyte with excellent tolerance to salt. Previous studies have revealed certain physiological mechanisms and detected functional genes associated with salt tolerance. However, knowledge on the ROS scavenging system and regulatory mechanisms in this species when adapting to salinity is limited. Therefore, this study further analyzed the transcriptional changes in genes related to the ROS scavenging system and important regulatory mechanisms in A. canescens under saline conditions using our previous RNA sequencing data. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation revealed that the differentially expressed genes (DEGs) were highly enriched in signal transduction- and reactive oxygen species-related biological processes, including "response to oxidative stress", "oxidoreductase activity", "protein kinase activity", "transcription factor activity", and "plant hormone signal transduction". Further analyses suggested that the transcription abundance of many genes involved in SOD, the AsA-GSH cycle, the GPX pathway, PrxR/Trx, and the flavonoid biosynthesis pathway were obviously enhanced. These pathways are favorable for scavenging excessive ROS induced by salt and maintaining the integrity of the cell membrane. Meanwhile, many vital transcription factor genes (WRKY, MYB, ZF, HSF, DREB, and NAC) exhibited increased transcripts, which is conducive to dealing with saline conditions by regulating downstream salt-responsive genes. Furthermore, a larger number of genes encoding protein kinases (RLK, CDPK, MAPK, and CTR1) were significantly induced by saline conditions, which is beneficial to the reception/transduction of salt-related signals. This study describes the abundant genetic resources for enhancing the salt tolerance in salt-sensitive plants, especially in forages and crops.

Elucidation of the molecular mechanisms by which plants sense and respond to environmental stimuli that influence their growth and yield is a prerequisite for understanding the adaptation of plants to climate change. Plants are sessile organisms and one important factor for their successful acclimation is the temporal coordination of the 24 h daily cycles and the stress response. The crosstalk between second messengers, such as Ca2+, reactive oxygen species (ROS), and hormones is a fundamental aspect in plant adaptation and survival under environmental stresses. In this sense, the circadian clock, in conjunction with Ca2+- and hormone-signalling pathways, appears to act as an important mechanism controlling plant adaptation to stress. The relationship between the circadian clock and ROS-generating and ROS-scavenging mechanisms is still not fully understood, especially at the post-transcriptional level and in stress situations in which ROS levels increase and changes in cell redox state occur. In this review, we summarize the information regarding the relationship between the circadian clock and the ROS homeostasis network. We pay special attention not only to the transcriptional regulation of ROS-generating and ROS-scavenging enzymes, but also to the few studies that have been performed at the biochemical level and those conducted under stress conditions.

Plants are sessile organisms presenting different adaptation mechanisms that allow their survival under adverse situations. Among them, reactive oxygen and nitrogen species (ROS, RNS) and H₂S are emerging as components not only of cell development and differentiation but of signaling pathways involved in the response to both biotic and abiotic attacks. The study of the posttranslational modifications (PTMs) of proteins produced by those signaling molecules is revealing a modulation on specific targets that are involved in many metabolic pathways in the different cell compartments. These modifications are able to translate the imbalance of the redox state caused by exposure to the stress situation in a cascade of responses that finally allow the plant to cope with the adverse condition. In this review we give a generalized vision of the production of ROS, RNS, and H₂S in plant mitochondria. We focus on how the principal mitochondrial processes mainly the electron transport chain, the tricarboxylic acid cycle and photorespiration are affected by PTMs on cysteine residues that are produced by the previously mentioned signaling molecules in the respiratory organelle. These PTMs include S-oxidation, S-glutathionylation, S-nitrosation, and persulfidation under normal and stress conditions. We pay special attention to the mitochondrial Thioredoxin/Peroxiredoxin system in terms of its oxidation-reduction posttranslational targets and its response to environmental stress.

Seeds enable plant survival in harsh environmental conditions, and via seeds, genetic information is transferred from parents to the new generation; this stage provides an opportunity for sessile plants to settle in new territories. However, seed viability decreases over long-term storage due to seed aging. For the effective conservation of gene resources, e.g., in gene banks, it is necessary to understand the causes of decreases in seed viability, not only where the aging process is initiated in seeds but also the sequence of events of this process. Mitochondria are the main source of reactive oxygen species (ROS) production, so they are more quickly and strongly exposed to oxidative damage than other organelles. The mitochondrial antioxidant system is also less active than the antioxidant systems of other organelles, thus such mitochondrial 'defects' can strongly affect various cell processes, including seed aging, which we discuss in this paper.

In a changing environment, plants are able to acclimate to new conditions by regulating their metabolism through the antioxidant and redox systems involved in the stress response. Here, we studied a mitochondrial thioredoxin in wild-type (WT) Arabidopis thaliana and two Attrxo1 mutant lines grown in the absence or presence of 100 mM NaCl. Compared to WT plants, no evident phenotype was observed in the mutant plants under control condition, although they had higher number of stomata, loss of water, nitric oxide and carbonyl protein contents as well as higher activity of superoxide dismutase (SOD) and catalase enzymes than WT plants. Under salinity, the mutants presented lower water loss and higher stomatal closure, H₂ O₂ and lipid peroxidation levels accompanied by higher enzymatic activity of catalase and the different SOD isoenzymes compared to WT plants. These inductions may collaborate in the maintenance of plant integrity and growth observed under saline conditions, possibly as a way to compensate the lack of TRXo1. We discuss the potential of TRXo1 to influence the development of the whole plant under saline conditions, which have great value for the agronomy of plants growing under unfavorable environment.

Mitochondrial thioredoxin-o (AtTrxo1) was characterized and its expression examined in different organs of Arabidopsis thaliana. AtTrxo1 transcript levels were particularly high in dry seeds and cotyledons where they reached a maximum 36 h after imbibition with water, coinciding with 50% germination. Expression was lower in seeds germinating in 100 mM NaCl. To gain insight into the transcriptional regulation of the AtTrxo1 gene, a phylogenomic analysis was coupled with the screening of an arrayed library of Arabidopsis transcription factors in yeast. The basic leucine zipper AtbZIP9 and the zinc finger protein AZF2 were identified as putative transcriptional regulators. Transcript regulation of AtbZIP9 and AtAFZ2 during germination was compatible with the proposed role in transcriptional regulation of AtTrxo1. Transient over-expression of AtbZIP9 and AtAZF2 in Nicotiana benthamiana leaves demonstrated an activation effect of AtbZIP9 and a repressor effect of AtAZF2 on AtTrxo1 promoter-driven reporter expression. Although moderate concentrations of salt delayed germination in Arabidopsis wild-type seeds, those of two different AtTrxo1 knock-out mutants germinated faster and accumulated higher H2O2 levels than the wild-type. All these data indicate that AtTrxo1 has a role in redox homeostasis during seed germination under salt conditions.

Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys¹⁷⁴), but not the peroxidatic equivalent (Cys⁵²), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys⁵⁹) and resolving (Cys⁸⁴) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins.

A type II peroxiredoxin gene (XvPrx2) was isolated from a Xerophyta viscosa (Baker) cDNA cold-stress library. The polypeptide displayed significant similarity to other plant type II peroxiredoxins, with the conserved amino acid motif (PGAFTPTCS) proposed to constitute the active site of the enzyme. Northern blot analyses showed that XvPrx2 gene was stress-inducible in response to abiotic stresses while gel analyses revealed that XvPrx2 homologues exist within the X. viscosa proteome. Using a yellow fluorescent reporter protein, the XvPrx2 protein localised to the cytosol. A mutated protein (XvV7) was generated by converting the valine at position 76 to a cysteine and an in vitro DNA protection assay showed that, in the presence of either XvPrx2 or XvV7, DNA protection occurred. In addition, an in vivo assay showed that increased protection was conferred to Escherichia coli cells overexpressing either XvPrx2 or XvV7. The XvPrx2 activity was maximal with DTT as electron donor and H2O2 as substrate. Using E. coli thioredoxin, a 2-15-fold lower enzyme activity was observed. The XvPrx2 activity with glutathione was significantly lower and glutaredoxin had no measurable effect on this reaction. The XvV7 protein displayed significantly lower activity compared with XvPrx2 for all substrates assessed.

Reactive oxygen species (ROS) have a profound influence on almost every aspect of plant biology. Here, we emphasize the fundamental, intimate relationships between light-driven reductant formation, ROS, and oxidative stress, together with compartment-specific differences in redox buffering and the perspectives for their analysis. Calculations of approximate H2 O2 concentrations in the peroxisomes are provided, and based on the likely values in other locations such as chloroplasts, we conclude that much of the H2 O2 detected in conventional in vitro assays is likely to be extracellular. Within the context of scant information on ROS perception mechanisms, we consider current knowledge, including possible parallels with emerging information on oxygen sensing. Although ROS can sometimes be signals for cell death, we consider that an equally important role is to transmit information from metabolism to allow appropriate cellular responses to developmental and environmental changes. Our discussion speculates on novel sensing mechanisms by which this could happen and how ROS could be counted by the cell, possibly as a means of monitoring metabolic flux. Throughout, we place emphasis on the positive effects of ROS, predicting that in the coming decades they will increasingly be defined as hallmarks of viability within a changing and challenging environment.

To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.

Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.

Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis.

AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.

In plants, the presence of thioredoxin (Trx), peroxiredoxin (Prx), and sulfiredoxin (Srx) has been reported as a component of a redox system involved in the control of dithiol-disulfide exchanges of target proteins, which modulate redox signalling during development and stress adaptation. Plant thiols, and specifically redox state and regulation of thiol groups of cysteinyl residues in proteins and transcription factors, are emerging as key components in the plant response to almost all stress conditions. They function in both redox sensing and signal transduction pathways. Scarce information exists on the transcriptional regulation of genes encoding Trx/Prx and on the transcriptional and post-transcriptional control exercised by these proteins on their putative targets. As another point of control, post-translational regulation of the proteins, such as S-nitrosylation and S-oxidation, is of increasing interest for its effect on protein structure and function. Special attention is given to the involvement of the Trx/Prx/Srx system and its redox state in plant signalling under stress, more specifically under abiotic stress conditions, as an important cue that influences plant yield and growth. This review focuses on the regulation of Trx and Prx through cysteine S-oxidation and/or S-nitrosylation, which affects their functionality. Some examples of redox regulation of transcription factors and Trx- and Prx-related genes are also presented.

Peroxiredoxins (Prxs) have emerged as important factors linking reactive oxygen species (ROS) metabolism to redox-dependent signaling events. Together with ROS, nitric oxide (NO) is a free radical product of the cell metabolism that is essential in the signal transduction. S-Nitrosylation is emerging as a fundamental protein modification for the transduction of NO bioactivity. Using recombinant pea mitochondrial PsPrxII F (PrxII F), the effect of S-nitrosoglutathione (GSNO) and sodium nitroprusside dehydrate (SNP), which are known to mediate protein S-nitrosylation processes, was studied. S-Nitrosylation of the PrxII F was demonstrated using the biotin switch method and LC ESI-QTOF tandem MS analysis. S-nitrosylated PrxII F decreased its peroxidase activity and acquired a new transnitrosylase activity, preventing the thermal aggregation of citrate synthase (CS). For the first time, we demonstrate the dual function for PrxII F as peroxidase and transnitrosylase. This switch was accompanied by a conformational change of the protein that could favor the protein-protein interaction CS-PrxII F. The observed in vivo S-nitrosylation of PrxII F could probably function as a protective mechanism under oxidative and nitrosative stress, such as occurs under salinity. We conclude that we are dealing with a novel regulatory mechanism for this protein by NO.

S-Nitrosylation is a post-translational modification that is increasingly viewed as fundamental for the signal transduction role of NO in plants. This study demonstrates that S-nitrosylation of the mitochondrial peroxiredoxin PrxII F induces a conformational change in the protein and provokes a reduction in its peroxidase activity, while acquiring a novel function as transnitrosylase. The implication of this mechanism will increase our understanding of the role of posttranslational modifications in the protein function in plants under stress situations such as salinity, in which NO could act as signaling molecule.

Oxidative stress resulting from increased availability of reactive oxygen species (ROS) is a key component of many responses of plants to challenging environmental conditions. The consequences for plant metabolism are complex and manifold. We review data on small compounds involved in oxidative stress, including ROS themselves and antioxidants and redox buffers in the membrane and soluble phases, and we discuss the wider consequences for plant primary and secondary metabolism. While metabolomics has been exploited in many studies on stress, there have been relatively few non-targeted studies focused on how metabolite signatures respond specifically to oxidative stress. As part of the discussion, we present results and reanalyze published datasets on metabolite profiles in catalase-deficient plants, which can be considered to be model oxidative stress systems. We emphasize the roles of ROS-triggered changes in metabolites as potential oxidative signals, and discuss responses that might be useful as markers for oxidative stress. Particular attention is paid to lipid-derived compounds, the status of antioxidants and antioxidant breakdown products, altered metabolism of amino acids, and the roles of phytohormone pathways.

A 2-Cys peroxiredoxin cDNA (CjPrx) was isolated and characterized from Caragana jubata, a temperate/alpine plant species of high altitude cold desert of Himalaya and Eurasia. The cDNA obtained was 1,064 bp long consisting of an open reading frame of 789 bp encoding 262 amino acids. The calculated molecular mass of the mature protein was 28.88 kDa and pI was 5.84. Deduced amino acid sequence of CjPrx shared a high degree homology with 2-CysPrx proteins from other plants. CjPrx had both the PRX_type 2-Cys domain and thioredoxin-like superfamily domains. CjPrx contained 26.72% α-helices, 6.87% β-turns, 20.61% extended strands and 45.80% random coils, and was a hydrophilic protein. Expression of CjPrx was modulated by low temperature, methyl jasmonate (MJ), salicylic acid and drought stress, but no significant change was observed in response to abscisic acid treatment. Among all the treatments, a strong up-regulation of CjPrx was observed in response to MJ treatment.

As a result of the industrial revolution, anthropogenic activities have enhanced there distribution of many toxic heavy metals from the earth's crust to different environmental compartments. Environmental pollution by toxic heavy metals is increasing worldwide, and poses a rising threat to both the environment and to human health.Plants are exposed to heavy metals from various sources: mining and refining of ores, fertilizer and pesticide applications, battery chemicals, disposal of solid wastes(including sewage sludge), irrigation with wastewater, vehicular exhaust emissions and adjacent industrial activity.Heavy metals induce various morphological, physiological, and biochemical dysfunctions in plants, either directly or indirectly, and cause various damaging effects. The most frequently documented and earliest consequence of heavy metal toxicity in plants cells is the overproduction of ROS. Unlike redox-active metals such as iron and copper, heavy metals (e.g, Pb, Cd, Ni, AI, Mn and Zn) cannot generate ROS directly by participating in biological redox reactions such as Haber Weiss/Fenton reactions. However, these metals induce ROS generation via different indirect mechanisms, such as stimulating the activity of NADPH oxidases, displacing essential cations from specific binding sites of enzymes and inhibiting enzymatic activities from their affinity for -SH groups on the enzyme.Under normal conditions, ROS play several essential roles in regulating the expression of different genes. Reactive oxygen species control numerous processes like the cell cycle, plant growth, abiotic stress responses, systemic signalling, programmed cell death, pathogen defence and development. Enhanced generation of these species from heavy metal toxicity deteriorates the intrinsic antioxidant defense system of cells, and causes oxidative stress. Cells with oxidative stress display various chemical,biological and physiological toxic symptoms as a result of the interaction between ROS and biomolecules. Heavy-metal-induced ROS cause lipid peroxidation, membrane dismantling and damage to DNA, protein and carbohydrates. Plants have very well-organized defense systems, consisting of enzymatic and non-enzymatic antioxidation processes. The primary defense mechanism for heavy metal detoxification is the reduced absorption of these metals into plants or their sequestration in root cells.Secondary heavy metal tolerance mechanisms include activation of antioxidant enzymes and the binding of heavy metals by phytochelatins, glutathione and amino acids. These defense systems work in combination to manage the cascades of oxidative stress and to defend plant cells from the toxic effects of ROS.In this review, we summarized the biochemiCal processes involved in the over production of ROS as an aftermath to heavy metal exposure. We also described the ROS scavenging process that is associated with the antioxidant defense machinery.Despite considerable progress in understanding the biochemistry of ROS overproduction and scavenging, we still lack in-depth studies on the parameters associated with heavy metal exclusion and tolerance capacity of plants. For example, data about the role of glutathione-glutaredoxin-thioredoxin system in ROS detoxification in plant cells are scarce. Moreover, how ROS mediate glutathionylation (redox signalling)is still not completely understood. Similarly, induction of glutathione and phytochelatins under oxidative stress is very well reported, but it is still unexplained that some studied compounds are not involved in the detoxification mechanisms. Moreover,although the role of metal transporters and gene expression is well established for a few metals and plants, much more research is needed. Eventually, when results for more metals and plants are available, the mechanism of the biochemical and genetic basis of heavy metal detoxification in plants will be better understood. Moreover, by using recently developed genetic and biotechnological tools it may be possible to produce plants that have traits desirable for imparting heavy metal tolerance.

Glutathione is a small redox-active molecule existing in two main stable forms: the thiol (GSH) and the disulphide (GSSG). In plants growing in optimal conditions, the GSH:GSSG ratio is high in most cell compartments. Challenging environmental conditions are known to alter this ratio, notably by inducing the accumulation of GSSG, an effect that may be influential in the perception or transduction of stress signals. Despite the potential importance of glutathione status in redox signaling, the reactions responsible for the oxidation of GSH to GSSG have not been clearly identified. Most attention has focused on the ascorbate-glutathione pathway, but several other candidate pathways may couple the availability of oxidants such as H2O2 to changes in glutathione and thus impact on signaling pathways through regulation of protein thiol-disulfide status. We provide an overview of the main candidate pathways and discuss the available biochemical, transcriptomic, and genetic evidence relating to each. Our analysis emphasizes how much is still to be elucidated on this question, which is likely important for a full understanding of how stress-related redox regulation might impinge on phytohormone-related and other signaling pathways in plants.

Norway maple (Acer platanoides L., orthodox) and sycamore (Acer pseudoplatanus L., recalcitrant) belong to the same genus and grow under similar climatic conditions, but their seeds differ in their tolerance to desiccation. The initial water content (WC) of the seeds used in this study was 50%, and they were dried to 40, 20 and 7%. The mitochondrial peroxiredoxin IIF (PRXIIF) was identified in seeds of both species by immunoblotting. Semiquantitative RT-PCR analyses indicated that the transcript level of PRXIIF in both types of seeds increased during different stages of desiccation and was higher in seeds of Norway maple than in sycamore. General proteome analyses showed important differences between orthodox and recalcitrant seeds. In sycamore seeds that had been desiccated to a 7% WC, the number of protein spots and the levels of those spots were lower than in desiccation-tolerant Norway maple seeds. Post-translational modifications of PRXIIF in seeds at a 50% WC were detected via 2D electrophoresis and subsequent western blot analysis. The detected shift in the pI values (± 0.3) in A. pseudoplatanus was possibly caused by phosphorylation because several potential phosphorylation sites were predicted in silico for that protein. The gene and amino acid sequences were obtained and aligned with known sequences of other plant PRXIIF genes and proteins. High values of sequence identity were noted between the PRXIIF protein sequences of Acer species, Populus trichocarpa Torr. & A. Gray and Arabidopsis thaliana (L.) Heynh. The involvement of PRXIIF in defining the physiological differences between desiccation-tolerant and desiccation-sensitive Acer seeds is discussed in the context of its role in mitochondrial redox homeostasis.

For a plant to grow and develop, energy and appropriate building blocks are a fundamental requirement. Mitochondrial respiration is a vital source for both. The delicate redox processes that make up respiration are affected by the plant's changing environment. Therefore, mitochondrial regulation is critically important to maintain cellular homeostasis. This involves sensing signals from changes in mitochondrial physiology, transducing this information, and mounting tailored responses, by either adjusting mitochondrial and cellular functions directly or reprogramming gene expression.

Retrograde (RTG) signaling, by which mitochondrial signals control nuclear gene expression, has been a field of very active research in recent years. Nevertheless, no mitochondrial RTG-signaling pathway is yet understood in plants. This review summarizes recent advances toward elucidating redox processes and other bioenergetic factors as a part of RTG signaling of plant mitochondria.

Novel insights into mitochondrial physiology and redox-regulation provide a framework of upstream signaling. On the other end, downstream responses to modified mitochondrial function have become available, including transcriptomic data and mitochondrial phenotypes, revealing processes in the plant that are under mitochondrial control.

Drawing parallels to chloroplast signaling and mitochondrial signaling in animal systems allows to bridge gaps in the current understanding and to deduce promising directions for future research. It is proposed that targeted usage of new technical approaches, such as quantitative in vivo imaging, will provide novel leverage to the dissection of plant mitochondrial signaling.

Together with reactive oxygen species, nitric oxide is an essential part of the signal transduction induced by stress conditions. In this work we study the pattern of S-nitrosylated proteins from mitochondria of pea plants subjected to 150mM NaCl for 5 and 14days. A differential pattern of target proteins was found during plant development and salt stress, with a minor number of S-nitrosylated proteins at 14 days specifically some key enzymes related to respiration and photorespiration. At this time of stress, only ATP synthase β subunit, peroxiredoxin and Hsp90 were S-nitrosylated and no changes in protein levels were observed, although the activity of PrxII F may be reduced by S-nitrosylation. The NADH/NAD(+) ratio was also high at 14days but not the NADPH/NADP(+). An enhancement in NO measured by fluorimetry and confocal microscopy was observed in leaves, being part of the NO localized in mitochondria. An increase in mitochondrial GSNOR activity was produced in response to short and long-term NaCl treatment, where a higher number of nitrated proteins were also observed. The results indicated that posttranslational modifications seem to modulate respiratory and photorespiratory pathways, as well as some antioxidant enzymes, through differential S-nitrosylation/denitrosylation in control conditions and under salt stress.

Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).

Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.

Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx.

The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.

Mitochondria play an essential role in reactive oxygen species (ROS) signal transduction in plants. Redox regulation is an essential feature of mitochondrial function, with thioredoxin (Trx), involved in disulphide/dithiol interchange, playing a prominent role. To explore the participation of mitochondrial PsTrxo1, Mn-superoxide dismutase (Mn-SOD), peroxiredoxin (PsPrxII F), and alternative oxidase (AOX) under salt stress, their transcriptional and protein levels were analysed in pea plants growing under 150 mM NaCl for a short and a long period. The activities of mitochondrial Mn-SOD and Trx together with the in vivo activities of the alternative pathway (AP) and the cytochrome pathway (CP) were also determined, combined with the characterization of the plant physiological status as well as the mitochondrial oxidative indicators. The analysis of protein and mRNA levels and activities revealed the importance of the post-transcriptional and post-translational regulation of these proteins in the response to salt stress. Increases in AOX protein amount correlated with increases in AP capacity, whereas in vivo AP activity was maintained under salt stress. Similarly, Mn-SOD activity was also maintained. Under all the stress treatments, photosynthesis, stomatal conductance, and CP activity were decreased although the oxidative stress in leaves was only moderate. However, an increase in lipid peroxidation and protein oxidation was found in mitochondria isolated from leaves under the short-term salinity conditions. In addition, an increase in mitochondrial Trx activity was produced in response to the long-term NaCl treatment. The results support a role for PsTrxo1 as a component of the defence system induced by NaCl in pea mitochondria, providing the cell with a mechanism by which it can respond to changing environment protecting mitochondria from oxidative stress together with Mn-SOD, AOX, and PrxII F.

Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

Sulfiredoxin (Srx) couples the energy of ATP hydrolysis to the energetically unfavorable process of reducing the inactive sulfinic form of 2-cysteine peroxiredoxins (Prxs) to regenerate its active form. In plants, Srx as well as typical 2-cysteine Prx have been considered as enzymes with exclusive chloroplast localization. This work explores the subcellular localization of Srx in pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana). Immunocytochemistry, analysis of protein extracts from isolated intact organelles, and cell-free posttranslational import assays demonstrated that plant Srx also localizes to the mitochondrion in addition to plastids. The dual localization was in line with the prediction of a signal peptide for dual targeting. Activity tests and microcalorimetric data proved the interaction between Srx and its mitochondrial targets Prx IIF and thioredoxin. Srx catalyzed the retroreduction of the inactive sulfinic form of atypical Prx IIF using thioredoxin as reducing agent. Arabidopsis Srx also reduced overoxidized human Prx V. These results suggest that plant Srx could play a crucial role in the regulation of Prx IIF activity by controlling the regeneration of its overoxidized form in mitochondria, which are sites of efficient reactive oxygen species production in plants.

The largest group of plant thioredoxins (TRXs) consists of the so-called h-type; their great number raises questions about their specific or redundant roles in plant cells. Pisum sativum thioredoxin h1 (PsTRXh1) and Pisum sativum thioredoxin h2 (PsTRXh2) are both h-type TRXs from pea (Pisum sativum) previously identified and biochemically characterized. While both are involved in redox regulation and show a high-sequence identity (60%), they display different behavior during in vitro and in vivo assays. In this work, we show that these two proteins display different specificity in the capturing of protein targets in vitro, by the use of a new stringent method. PsTRXh2 interacted with classical antioxidant proteins, whereas PsTRXh1 showed a completely different pattern of targeted proteins, and was able to capture a transcription factor. We also showed that the two proteins display very different thermal and chemical stabilities. We suggest that the differences in thermal and chemical stability point to a distinct and characteristic pattern of protein specificity.

Peroxiredoxins (Prx) are ubiquitous thiol-dependent peroxidases capable of reducing a broad range of toxic peroxides and peroxinitrites. A cysteinyl residue of peroxiredoxins reacts with the peroxides as primary catalytic center and oxidizes to sulfenic acid. The regeneration of the reduced form of Prx is required as a next step to allow its entry into next catalytic cycle. Several proteins, such as thioredoxin, glutaredoxin, cyclophilin, among others, are known to facilitate the regeneration of the reduced (catalytically active) form of Prx in plants. Based on the cysteine residues conserved in the deduced amino acid sequence and their catalytic mechanisms, four groups of peroxiredoxins have been distinguished in plants, namely, 1-Cys Prx, 2-Cys Prx, Type II Prx and Prx Q. Peroxiredoxins are known to play an important role in combating the reactive oxygen species generated at the level of electron transport activities in the plant exposed to different types of biotic and abiotic stresses. In addition to their role in antioxidant defense mechanisms in plants, they also modulate redox signaling during development and adaptation. Besides these general properties, peroxiredoxins have been shown to protect DNA from damage in vitro and in vivo. They also regulate metabolism in thylakoids and mitochondria. The present review summarizes the most updated information on the structure and catalysis of Prx and their functional importance in plant metabolism.

Isothermal titration calorimetry (ITC) is a powerful technique for investigating self-association processes of protein complexes and was expected to reveal quantitative data on peroxiredoxin oligomerization by directly measuring the thermodynamic parameters of dimer-dimer interaction. Recombinant classical 2-cysteine peroxoredoxins from Homo sapiens, Arabidopsis thaliana, and Pisum sativum as well as a carboxy-terminally truncated variant were subjected to ITC analysis by stepwise injection into the reaction vessel under various redox conditions. The direct measurement of the decamer-dimer equilibrium of reduced peroxiredoxin revealed a critical concentration in the very low micromolar range. The data suggest a cooperative assembly above this critical transition concentration where a nucleus facilitates assembly. The rather abrupt transition indicates that assembly processes do not occur below the critical transition concentration while oligomerization is efficiently triggered above it. The magnitude of the measured enthalpy confirmed the endothermic nature of the peroxiredoxin oligomerization. Heterocomplexes between peroxiredoxin polypeptides from different species were not formed. We conclude that a functional constraint conserved the dimer-decamer transition with highly similar critical transition concentrations despite emerging sequence variation during evolution.

Peroxiredoxins (Prxs) are ubiquitous thiol-dependent peroxidases capable of eliminating a variety of peroxides through reactive catalytic cysteines, which are regenerated by reducing systems. Based on amino acid sequences and their mode of catalysis, five groups of thiol peroxidases have been distinguished in plants, and type II Prx is one of them with representatives in many sub-cellular compartments. The mature form of poplar chloroplastic Prx IIE was expressed as a recombinant protein in Escherichia coli. The protein is able to reduce H2O2 and tert-butyl hydroperoxide and is regenerated by both glutaredoxin (Grx) and thioredoxin (Trx) systems. Nevertheless, compared with Trxs, Grxs, and more especially chloroplastic Grx S12, are far more efficient reductants towards Prx IIE. The expression of Prx IIE at both the mRNA and protein levels as a function of organ type and abiotic stress conditions was investigated. Western blot analysis revealed that Prx IIE gene is constitutively expressed in Arabidopsis thaliana, mostly in young and mature leaves and in flowers. Under photo-oxidative treatment and water deficit, almost no change was observed in the abundance of Prx IIE in A. thaliana, while the level of Prx Q (one of the two other chloroplastic Prxs with 2-Cys Prx) increased in response to both stresses, indicating that plastidic members of the Prx family exhibit specific patterns of expression under stress.