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The history of the series is described by three successive Editors. Each one reports how he has been approached, his conception of the different volumes and gives an overview of the topics which have been treated. The first period, from creation of the series in 1963 by Professor Reginald Preston to 2006, was covered by Professor James (Jim) Callow who became Editor after Professor Harold Woolhouse, in 1985. During this period, 44 volumes were published, essentially eclectic, covering most of the fields in plant biology. Under James Callow's editorship, the frequency of issues increased up to two/year and a few thematic volumes were published. When he decided to stop in 2006, Dr. Jean Claude Kader and Dr. Michel Delseny were appointed. They maintained a rate of two to three issues/year, alternating eclectic and thematic volumes. They together published 16 volumes and resigned in 2012. Finally, they were replaced by Professors Jean-Pierre Jacquot and Pierre Gadal. They published only thematic volumes and the rate increased to four–five issues/year.

Volume 52 of Advances in Botanical Research “Oxidative stress and redox regulation in plants” edited by Jean-Pierre Jacquot (series Editors Michel Delseny, Jean-Claude Kader) was published in 2009, so now more than 12 years ago. It featured 15 different chapters and more than 60 authors contributed. Volume 52 has covered many aspects of plant physiology including the generation of oxidative stress, the molecular responses to oxidative stress and redox regulation. In general such a volume is initiated about 2 years before final publication and so it has been now 15 years overall. We believe thus that it is now appropriate for the anniversary volume 100 of ABR to summarize what was described in that volume but also to provide a survey of more recent literature showing how the plant redox field has evolved and expanded since.

The special issue on Thioredoxin and Glutaredoxin systems (https://www.mdpi.com/journal/antioxidants/special_issues/Thioredoxin_and_Glutaredoxin_Systems) was initiated in response to solicitations from Antioxidants after discussing with colleagues at two successive redox meetings sponsored by European Molecular Biology Organization (EMBO) and held in July 2017 in Moscow/St. Petersburg (http://redox.vub.ac.be/events/embo-redox-biology-conference.html) and in September of the same year in San Feliu de Guixols (Spain) (http://meetings.embo.org/event/17-thiol-ox).[br/] We could then submit the idea to long time collaborators and redox friends but also to other colleagues with whom we had the chance to get in touch with at these meetings. In general, although Antioxidants is a rather recent creation and its credentials were at the time not so well known, the idea of participating in a special issue was very well received and many of the contacted colleagues have answered positively.[br/] Of course, as our background is in plant sciences, this special issue mostly contains papers dealing with oxygenic phototrophs but other experimental model organisms are also addressed (bacteria, mammals, zebrafish, etc.). Overall the special issue contains 16 papers, 12 of those reporting experimental research data, and 4 others being more review-like although some of them also contain original bioinformatics data.

Thiol-based redox regulation is central to adjusting chloroplast functions under varying light conditions. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been well recognized to mediate the light-responsive reductive control of target proteins; however, the molecular basis for reoxidizing its targets in the dark remains unidentified. Here, we report a mechanism of oxidative thiol modulation in chloroplasts. We biochemically characterized a chloroplast stroma-localized atypical Trx from Arabidopsis, designated as Trx-like2 (TrxL2). TrxL2 had redox-active properties with an unusually less negative redox potential. By an affinity chromatography-based method, TrxL2 was shown to interact with a range of chloroplast redox-regulated proteins. The direct discrimination of thiol status indicated that TrxL2 can efficiently oxidize, but not reduce, these proteins. A notable exception was found in 2-Cys peroxiredoxin (2CP); TrxL2 was able to reduce 2CP with high efficiency. We achieved a complete in vitro reconstitution of the TrxL2/2CP redox cascade for oxidizing redox-regulated proteins and draining reducing power to hydrogen peroxide (H(2)O(2)). We further addressed the physiological relevance of this system by analyzing protein-oxidation dynamics. In Arabidopsis plants, a decreased level of 2CP led to the impairment of the reoxidation of redox-regulated proteins during light–dark transitions. A delayed response of protein reoxidation was concomitant with the prolonged accumulation of reducing power in TrxL2. These results suggest an in vivo function of the TrxL2/2CP redox cascade for driving oxidative thiol modulation in chloroplasts.

Redox regulation of chloroplast enzymes via disulphide reduction is believed to control the rates of CO 2 fixation. The study of the thioredoxin reduction pathways and of various target enzymes lead to the following guidelines: i) Thioredoxin gene content is greatly higher in photosynthetic eukaryotes compared to prokaryotes; ii) Thioredoxin-reducing pathways have expanded in photosynthetic eukaryotes with four different thioredoxin reductases and the possibility to reduce some thioredoxins via glutaredoxins; iii) Some enzymes that were thought to be strictly linked to photosynthesis ferredoxin-thioredoxin reductase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase, sedoheptulose-1,7-bisphosphatase are present in non-photosynthetic organisms; iv) Photosynthetic eukaryotes contain a genetic patchwork of sequences borrowed from prokaryotes including α–proteobacteria and archaea; v) The introduction of redox regulatory sequences did not occur at the same place for all targets. Some possess critical cysteines in cyanobacteria, for others the transition occurred rather at the green algae level; vi) Generally the regulatory sites of the target enzymes are distally located from the catalytic sites. The cysteine residues are generally not involved in catalysis. Following reduction, molecular movements open the active sites and make catalysis possible; vii) The regulatory sequences are located on surface-accessible loops. At least one instance they can be cut out and serve as signal peptides for inducing plant defence.

International audience; The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatorymembers of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redoxactive disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg2+ and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.

Key message This paper describes how the combination of genomics, genetic engineering, and 3D structural characterization has helped clarify the redox regulatory networks in poplar with consequences not only in system biology in plants but also in bacteria and mammalian systems.[br/] Context Tree genomes are increasingly available with a large number of orphan genes coding for proteins, the function of which is still unknown.[br/] Aims and methods Modern techniques of genome analysis coupled with recombinant protein technology and massive 3D structural determination of tree proteins should help elucidate the function of many of the proteins encoded by orphan genes. X-ray crystallography and NMR will be the methods of choice for protein structure determination.[br/] Results In this review, we provide examples illustrating how the above-mentioned techniques improved our understanding of redox regulatory circuits in poplar, the first forest tree species sequenced. We showed that poplar peroxiredoxins use either thioredoxin or glutaredoxin as electron donors to reduce hydrogen peroxide. That glutaredoxin could be a reductant was unknown at the time of this discovery even in other biological organisms and was later confirmed notably by the observation that the two genes are fused in some bacteria and by the resolution of the structure of the bacterial hybrid protein. Similarly, genome analysis coupled to in vitro analysis of enzymatic properties led to the discovery that some plant methionine sulfoxide reductases can also use both thioredoxins and glutaredoxins as electron donors. Besides their disulfide reductase activity, it has been demonstrated that some poplar glutaredoxins are also involved in iron-sulfur center biogenesis and assembly. The original 3D structure determination has been made with poplar glutaredoxin C1 and then confirmed in a variety of other biological organisms including human. Our work also showed that in plants, so-called glutathione peroxidases use thioredoxins and not glutathione as electron donors. This is true for all non-selenocysteine-containing glutathione peroxidases. Finally, connections between the thioredoxin and glutaredoxin systems have been elucidated through the study of atypical poplar thioredoxins.[br/] Conclusion Altogether, these data illustrate how the combination of genetic engineering and structural biology improves our understanding of biological processes and helps fuel systems biology for trees and other biological species.

The validation of formal specifications is a challenging task. It is one of the factors that impede the penetration of formal methods into the common practices of software development. This paper discusses the issue of validating formal models by executing them in the context of Event-B. The most important problem lies in the non-determinism which often prevents purely automatic tools to execute models. In this paper, we first present and discuss the techniques we have created to allow the execution of models at all levels of abstraction. These techniques rely on users to overcome the barriers resulting from non-deterministic features by either modifying the model or providing ad hoc implementations. Then, we present our main contribution, the formal definition of the notion of fidelity, that guarantees that all the observable behaviors of the executable models are indeed specified by the original (non-deterministic) models. The notion of fidelity can be expressed in terms of proof obligations.

Key message Based on their impact on many ecosystems, we review the relevance of mosses in research regarding stress tolerance, metabolism, and cell biology. We introduce the potential use of mosses as complementary model systems in molecular forest research, with an emphasis on the most developed model mossPhyscomitrella patens. Context and aims Mosses are important components of several ecosystems. The moss P. patens is a well-established non-vascular model plant with a high amenability to molecular biology techniques and was designated as a JGI plant flagship genome. In this review, we will provide an introduction to moss research and highlight the characteristics of P. patens and other mosses as a potential complementary model system for forest research. Methods Starting with an introduction into general moss biology, we summarize the knowledge about moss physiology and differences to seed plants. We provide an overview of the current research areas utilizing mosses, pinpointing potential links to tree biology. To complement literature review, we discuss moss advantages and available resources regarding molecular biology techniques. Results and conclusion During the last decade, many fundamental processes and cell mechanisms have been studied in mosses and seed plants, increasing our knowledge of plant evolution. Additionally, moss-specific mechanisms of stress tolerance are under investigation to understand their resilience in ecosystems. Thus, using the advantages of model mosses such as P. patens is of high interest for various research approaches, including stress tolerance, organelle biology, cell polarity, and secondary metabolism.

A cDNA coding for a plastidic P2-type G6PDH isoform from A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants. (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRXs) was then investigated. Five cysteine to serine variants (C145S - C175S - C183S - C195S - C242S) and a variant with a double substitution for Cys175 and Cys183 (C175S/C183S) have been generated, purified and biochemically characterized in order to investigate the specific role(s) of cysteines in terms of redox regulation and NADPH-dependent inhibition. Three cysteine residues (C145, C194, C242) are suggested to have a role in controlling the NADP+ access to the active site, and in stabilizing the NADPH regulatory binding site. Our results also indicate that the regulatory disulfide involves residues Cys175 and Cys183 in a position similar to those of chloroplastic P1-G6PDHs, but the modulation is exerted primarily by TRX m-type, in contrast to P1-G6PDH, which is regulated by TRX f. This unexpected specificity indicates differences in the mechanism of regulation, and redox sensing of plastidic P2-G6PDH compared to chloroplastic P1-G6PDH in higher plants.

International audience; Glutaredoxins (Grxs) are oxidoreductases structurally related to thioredoxins (Trxs). They have two major proposed biochemical roles, the reduction of disulfide bonds and the binding of iron-sulfur (Fe-S) clusters, both of which usually involve glutathione. In photosynthetic organisms, Grxs are distributed into six classes. Several Grxs from classes I and II can indeed exist either as apoforms which display deglutathionylation activity or as holoforms which bind Fe-S clusters. Using biochemical, spectroscopic and structural approaches, we have characterized four plastidial Grxs, showing that three of these can bind an Fe-S cluster. Site-directed mutagenesis experiments and resolution of the crystal structure of class I Grxs (GrxC5 and S12) revealed the critical role of some active site residues for cluster formation and for protein activity. The deglutathionylation activity of these two Grxs proceeds through a monothiol mechanism, which is important for the regeneration of the thiol-peroxidase and methionine sulfoxide reductase families. The two plastidial class II Grxs (GrxS14 and S16) can bind more labile Fe-S clusters that can serve for the maturation of other proteins as demonstrated by in vitro Fe-S cluster transfer experiments and by the complementation of a yeast strain deleted for the mitochondrial Grx5. Using binary yeast two hybrid and bimolecular fluorescence complementation experiments, we have shown that class II but not class I Grxs interact with plastidial BolA proteins, including SufE1, a sulfurtransferase required for the maturation of plastidial Fe-S proteins. Finally, using spectroscopic and structural approaches, we have determined that the Grx-BolA couples form two types of complexes involving distinct regions of both partners. Hence, we propose (i) that, based on its peculiar catalytic and thermodynamic properties, GrxS12 and potentially GrxC5 could rather act as oxidoreductases and/or redox sensors, and (ii) that GrxS14 and GrxS16 could participate to the maturation of iron-sulfur proteins either by being directly involved in Fe-S cluster trafficking or by having regulatory roles in particular via their interaction with BolA proteins.

In plant cells, the plastidial glucose 6-phosphate dehydrogenase (P2-G6PDH, EC 1.1.1.49) represents one of the most important sources of NADPH. However, previous studies revealed that both native and recombinant purified P2-G6PDHs show a great instability and a rapid loss of catalytic activity. Therefore it has been difficult to describe accurately the catalytic and physico-chemical properties of these isoforms. The plastidial G6PDH encoding sequence from barley roots (Hordeum vulgare cv. Nure), devoid of a long plastidial transit peptide, was expressed as recombinant protein in Escherichia coil, either untagged or with an N-terminal his-tag. After purification from both the soluble fraction and inclusion bodies, we have explored its kinetic parameters, as well as its sensitivity to reduction. The obtained results are consistent with values determined for other P2-G6PDHs previously purified from barley roots and from other land plants. Overall, these data shed light on the catalytic mechanism of plant P2-G6PDH, summarized in a proposed model in which the sequential mechanism is very similar to the mammalian cytosolic G6PDH. This study provides a rational basis to consider the recombinant barley root P2-G6PDH as a good model for further kinetic and structural studies. (C) 2013 Elsevier Masson SAS. All rights reserved.

The Thermotoga maritima NADH:thioredoxin reductase (TmTR) contains FAD and a catalytic disulfide in the active center, and uses a relatively poorly studied physiological oxidant Grx-1-type glutaredoxin. In order to further assess the redox properties of TmTR, we used series of quinoidal and nitroaromatic oxidants with a wide range of single-electron reduction potentials (E-7(1), -0.49-0.09 V). We found that TmTR catalyzed the mixed single- and two-electron reduction of quinones and nitroaromatic compounds, which was much faster than the reduction of Grx-1. The reactivity of both groups of oxidants increased with an increase in their E-7(1), thus pointing to the absence of their structural specificity. The maximal rates of quinone reduction in the steady-state reactions were lower than the maximal rates of reduction of FAD by NADH, obtained in presteady-state experiments. The mixed-type reaction inhibition by NAD(+) was consistent with its competition for a NADH binding site in the oxidized enzyme form, and also with the reoxidation of the reduced enzyme form. The inhibition data yielded a value of the standard potential for TmTR of -0.31 +/- 0.03 V at pH 7.0, which may correspond to the FAD/FADH(2) redox couple. Overall, the mechanism of quinone- and nitroreductase reactions of T. maritima TR was similar to the previously described mechanism of Arabidopsis thaliana TR, and points to their prooxidant and possibly cytotoxic role.

Since their discovery in the soil bacterium Sorangium cellulosum, epothilones have emerged as a valuable substance class with promising anti-tumor activity. Because of their benefits in the treatment of cancer and neurodegenerative diseases, epothilones are targets for drug design and pharmaceutical research. The final step of their biosynthesis – a cytochrome P450 mediated epoxidation of epothilone C/D to A/B by CYP167A1 (EpoK) – needs significant improvement, in particular regarding the efficiency of its redox partners. Therefore, we have investigated the ability of various hetero- and homologous redox partners to transfer electrons to EpoK. Hereby, a new hybrid system was established with conversion rates eleven times higher and Vmax of more than seven orders of magnitudes higher as compared with the previously described spinach redox chain. This hybrid system is the most efficient redox chain for EpoK described to date. Furthermore, P450s from So ce56 were identified which are able to convert epothilone D to 14-OH, 21-OH, 26-OH epothilone D and 7-ketone epothilone D. The latter one represents a novel epothilone derivative and is a suitable candidate for pharmacological tests. The results revealed myxobacterial P450s from S. cellulosum So ce56 as promising candidates for protein engineering for biotechnological production of epothilone derivatives.

International audience; Glutaredoxins (Grxs) are oxidoreductases structurally related to thioredoxins (Trxs). They have two major proposed biochemical roles, the reduction of disulfide bonds and the binding of iron-sulfur (Fe-S) clusters, both of which usually involve glutathione. In photosynthetic organisms, Grxs are distributed into six classes. Several Grxs from classes I and II can indeed exist either as apoforms which display deglutathionylation activity or as holoforms which bind Fe-S clusters. Using biochemical, spectroscopic and structural approaches, we have characterized four plastidial Grxs, showing that three of these can bind an Fe-S cluster. Site-directed mutagenesis experiments and resolution of the crystal structure of class I Grxs (GrxC5 and S12) revealed the critical role of some active site residues for cluster formation and for protein activity. The deglutathionylation activity of these two Grxs proceeds through a monothiol mechanism, which is important for the regeneration of the thiol-peroxidase and methionine sulfoxide reductase families. The two plastidial class II Grxs (GrxS14 and S16) can bind more labile Fe-S clusters that can serve for the maturation of other proteins as demonstrated by in vitro Fe-S cluster transfer experiments and by the complementation of a yeast strain deleted for the mitochondrial Grx5. Using yeast two hybrid and bimolecular fluorescence complementation, we have shown that class II but not class I Grxs interact with plastidial BolA proteins, including SufE1, a sulfurtransferase required for the maturation of plastidial Fe-S proteins.Hence, we propose (i) that, based on its peculiar catalytic and thermodynamic properties, GrxS12 and potentially GrxC5 could act as redox sensors, and (ii) that GrxS14 and GrxS16 could participate to the maturation of iron-sulfur proteins either by being directly involved in Fe-S cluster trafficking or by having regulatory roles in particular via their interaction with BolA proteins.

International audience; A major hindrance to the use of formal methods is the difficulty to validate the models, particularly at the early stages of the development. We propose to build simulations: programs automatically generated from the specifications but with user-provided implementations of the non-executable traits of the models. We present such a simulation. Of course, the question of the fidelity of the simulation to the model is raised in such a setting. We provide a formal definition of fidelity and the proof obligations that can be attached to each hand-coded element so that fidelity can be proven.

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International audience; A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluorescent protein (GFP) fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic, and BolA4 dual-targeted to mitochondria and plastids. Binary yeast two-hybrid experiments demonstrated that all BolAs and SufE1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desulfurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionylated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several subcellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase ( P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.

International audience; The validation of formal models is a challenge for formal methods. We propose JeB, a framework which generates and executes simulations of Event-B models, even highly nondeterministic ones. JeB allows users to safely insert pieces of code to supply deterministic computations where the automatic translation fails. We present how JeB translates Event-B model into JavaScript. We define Fidelity as the formal notion which captures the idea of the correctness of a simulation. We define it through proof-obligations.

Photosynthetic organisms and especially terrestrial plants contain expanded redox regulatory pathways. Proteins involved in these redox chains and detoxification reactions often evolved on a thioredoxin fold structural basis. We discuss in this review the proteins associated with this superfamily, their modes of reduction/regeneration and their known protein targets, focusing in particular on the protein disulfide isomerase and glutathione transferase families. We also discuss the evolution of the redoxin and target families along the plant kingdom and provide information on the systems biology of the redox circuitry.

Three peroxiredoxins (Prxs) were identified in Thermotoga maritima, which possesses neither glutathione nor typical thioredoxins: one of the Prx6 class; one 2-Cys PrxBCP; and a unique hybrid protein containing an N-terminal 1-Cys PrxBCP domain fused to a flavin mononucleotide-containing nitroreductase (Ntr) domain. No peroxidase activity was detected for Prx6, whereas both bacterioferritin comigratory proteins (BCPs) were regenerated by a NADH/thioredoxin reductase/glutaredoxin (Grx)-like system, constituting a unique peroxide removal system. Only two of the three Grx-like proteins were able to support peroxidase activity. The inability of TmGrx1 to regenerate oxidized 2-Cys PrxBCP probably results from the thermodynamically unfavorable difference in their disulfide/dithiol Em values, −150 and −315 mV, respectively. Mutagenesis of the Prx-Ntr fusion, combined with kinetic and structural analyses, indicated that electrons are not transferred between its two domains. However, their separate activities could function in a complementary manner, with peroxide originating from the chromate reductase activity of the Ntr domain reduced by the Prx domain. Antioxid. Redox Signal. 18, 1613–1622.

International audience; Gravity perception and gravitropic response are essential for plant development. In herbaceous species, it is widely accepted that one of the primary events in gravity perception involves the displacement of amyloplasts within specialized cells. However, the early signaling events leading to stem reorientation are not fully known, especially in woody species in which primary and secondary growth occur. Thirty-six percent of the identified proteins that were differentially expressed after gravistimulation were established as potential Thioredoxin targets. In addition, Thioredoxin h expression was induced following gravistimulation. In situ immunolocalization indicated that Thioredoxin h protein co-localized with the amyloplasts located in the endodermal cells. These investigations suggest the involvement of Thioredoxin h in the first events of signal transduction in inclined poplar stems, leading to reaction wood formation.

Glutathione transferases (GSTs) are known to transfer glutathione onto small hydrophobic molecules in detoxification reactions. The GST Ure2pB1 from Phanerochaete chrysosporium exhibits atypical features, i.e. the presence of two glutathione binding sites and a high affinity towards oxidized glutathione. Moreover, PcUre2pB1 is able to efficiently deglutathionylate GS-phenacylacetophenone. Catalysis is not mediated by the cysteines of the protein but rather by the one of glutathione and an asparagine residue plays a key role in glutathione stabilization. Interestingly PcUre2pB1 interacts in vitro with a GST of the omega class. These properties are discussed in the physiological context of wood degrading fungi. Structured summary of protein interactions: PcUre2pB1 and PcUre2pB1 bind by X-ray crystallography (View interaction) PcUre2pB1 enzymaticly reacts PcGTO3 by enzymatic study (View interaction) (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Omega glutathione transferases (GSTO) constitute a family of proteins with variable distribution throughout living organisms. It is notably expanded in several fungi and particularly in the wood-degrading fungus Phanerochaete chlysosporium, raising questions concerning the function(s) and potential redundancy of these enzymes. Within the fungal families, GSTOs have been poorly studied and their functions remain rather sketchy. In this study, we have used fluorescent compounds as activity reporters to identify putative ligands. Experiments using 5-chloromethylfluorescein diacetate as a tool combined with mass analyses showed that GSTOs are able to cleave ester bonds. Using this property, we developed a specific activity-based profiling method for identifying ligands of PcGSTO3 and PcGSTO4. The results suggest that GSTOs could be involved in the catabolism of toxic compounds like tetralone derivatives. Biochemical investigations demonstrated that these enzymes are able to catalyze deglutathionylation reactions thanks to the presence of a catalytic cysteine residue. To access the physiological function of these enzymes and notably during the wood interaction, recombinant proteins have been immobilized on CNBr Sepharose and challenged with beech wood extracts. Coupled with GC-MS experiments this ligand fishing method allowed to identify terpenes as potential substrates of Omega GST suggesting a physiological role during the wood-fungus interactions. (C) 2012 Elsevier Masson SAS. All rights reserved.

Holo glutaredoxin (Grx) is a homo-dimer that bridges a [2Fe-2S] cluster with two glutathione (GSH) ligands. In this study, both monothiol and dithiol holo Grxs are found capable of transferring their iron-sulfur (FeS) cluster to an apo ferredoxin (Fdx) through direct interaction, regardless of FeS cluster stability in holo Grxs. The ligand GSH molecules in holo Grxs are unstable and can be exchanged with free GSH, which inhibits the FeS cluster transfer from holo Grxs to apo Fdx. This phenomenon suggests a novel role of GSH in FeS cluster trafficking.

Glutathione S-transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. Anew fungal specific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class in Phanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features by comparison with the canonical GST structure. An additional beta-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer and acts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements, including helix alpha 4' in the C-terminal domain, of the pre-sumed common ancestor of cytosolic GSTs (i.e. glutaredoxin 2). A sulfate binding site has been identified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid. Competition experiments between 8-anilino-1-naphtalene sulfonic acid, which has fluorescent properties, and various molecules showed that this GST binds glutathionylated and sulfated compounds but also wood extractive molecules, such as vanillin, chloronitrobenzoic acid, hydroxyacetophenone, catechins, and aldehydes, in the glutathione pocket. This enzyme could thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate, but alternatively and in a competitive way, it could also act as a ligandin of wood extractive compounds. These new structural and functional properties lead us to propose that this GST belongs to a new class that we name GSTFuA, for fungal specific GST class A.

Plant thioredoxins (Trxs) constitute a complex family of thiol oxidoreductases generally sharing a WCGPC active site sequence. Some recently identified plant Trxs (Clot, Trx-like1 and -2, Trx-lilium1, -2, and -3) display atypical active site sequences with altered residues between the two conserved cysteines. The transcript expression patterns, subcellular localizations, and biochemical properties of some representative poplar (Populus spp.) isoforms were investigated. Measurements of transcript levels for the 10 members in poplar organs indicate that most genes are constitutively expressed. Using transient expression of green fluorescent protein fusions, Clot and Trx-like1 were found to be mainly cytosolic, whereas Trx-like2.1 was located in plastids. All soluble recombinant proteins, except Clot, exhibited insulin reductase activity, although with variable efficiencies. Whereas Trx-like2.1 and Trx-lilium2.2 were efficiently regenerated both by NADPH-Trx reductase and glutathione, none of the proteins were reduced by the ferredoxin-Trx reductase. Only Trx-like2.1 supports the activity of plastidial thiol peroxidases and methionine sulfoxide reductases employing a single cysteine residue for catalysis and using a glutathione recycling system. The second active site cysteine of Trx-like2.1 is dispensable for this reaction, indicating that the protein possesses a glutaredoxin-like activity. Interestingly, the Trx-like2.1 active site replacement, from WCRKC to WCGPC, suppresses its capacity to use glutathione as a reductant but is sufficient to allow the regeneration of target proteins employing two cysteines for catalysis, indicating that the nature of the residues composing the active site sequence is crucial for substrate selectivity/recognition. This study provides another example of the cross talk existing between the glutathione/glutaredoxin and Trx-dependent pathways.

International audience; A natural fusion occurring between two tandemly repeated glutaredoxin (Grx) modules and a methionine sulfoxide reductase A (MsrA) has been detected in Gracilaria gracilis. Using an in vivo yeast complementation assay and in vitro activity measurements, we demonstrated that this fusion enzyme was able to reduce methionine sulfoxide into methionine using glutathione as a reductant. Consistently, a poplar cytosolic MsrA can be regenerated in vitro by glutaredoxins with an efficiency comparable to that of thioredoxins, but using a different mechanism. We hypothesize that the glutathione/glutaredoxin system could constitute an evolutionary conserved alternative regeneration system for MsrA.

Gpxs (glutathione peroxidases) constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms (SeCys-Gpx or Cys-Gpx), which are regenerated by glutathione or Trxs (thioredoxins) respectively. In the present paper we show new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. The present study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M−1·s−1. In PtGpx5 (poplar Gpx5; Pt is Populus trichocarpa), Glu79, which replaces the glutamine residue usually found in the Gpx catalytic tetrad, is likely to be involved in substrate selectivity. Although the redox midpoint potential of the Cys44–Cys92 disulfide bond and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide bond between the Trx catalytic cysteine residue and the Gpx5 resolving cysteine residue. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Also, MS analysis of in-vitro-oxidized PtGpx5 demonstrated that the peroxidatic cysteine residue can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen-peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.

International audience; Abstract--This paper explores the possibility to incorporate validation in the stepwise development process of formal specifications. Formal methods based on refinement break the intractable proof of the correctness of implementation into a sequence of many smaller proofs. Likewise, the validation of the specification could be broken into smaller steps associated to refinements with the technique of animation. Animating an abstract specification often requires to alter it in ways that proof obligations cannot be discharged anymore. So, we have developed a process and a set of transformation rules whose application produces an animatable specification which may be non-provable, but which is assured to have the same behavior. Guaranteeing behavioral preservation requires us to define an ad-hoc relationship between specifications based on a kind of trace semantics. 10 rules have been identified and proven to preserve behavior. Observations on the use of the technique on two case-studies are presented.

International audience; Abstract--In this paper, we explore the possibility to use Event-B as a formal domain modeling tool. We identify the areas where domain modelers can struggle and present some guidelines to avoid these pitfalls. We mainly address three questions about domain modeling: what to specify, how to refine, and how to verify. We discuss the strategy to express domain assumptions, protocols, time, and temporal properties. We also analyze the refinement and proof system of Event-B in this realm. We advocate small incremental steps and alternative refinement mechanisms, such as "observation levels." We find animation a very helpful activity to complement the verification process.

The glutathione-S-transferase (GST) proteins represent an extended family involved in detoxification processes. They are divided into various classes with high diversity in various organisms. The Ure2p class is especially expanded in saprophytic fungi compared to other fungi. This class is subdivided into two subclasses named Ure2pA and Ure2pB, which have rapidly diversified among fungal phyla. We have focused our analysis on Basidiomycetes and used Phanerochaete chrysosporium as a model to correlate the sequence diversity with the functional diversity of these glutathione transferases. The results show that among the nine isoforms found in P. chrysosporium, two belonging to Ure2pA subclass are exclusively expressed at the transcriptional level in presence of polycyclic aromatic compounds. Moreover, we have highlighted differential catalytic activities and substrate specificities between Ure2pA and Ure2pB isoforms. This diversity of sequence and function suggests that fungal Ure2p sequences have evolved rapidly in response to environmental constraints.

International audience; Ability to scale up from toy examples to real life problems is a crucial issue for formal methods. Formalizing a algorithm used in vehicle automation (platooning control) in a certification perspective, we had the opportunity to study the scaling up when going from a (toy) model in 1D to a (more realistic) model in 2D. The formalism, Event-B, belongs to the family of mathematical state based methods. Increase was quantitative: 3 times more events and 4 times more proofs; and qualitative: trigonometric functions and integrals are used. Edition and verification of the specification scale up well. The crucial part of the work was the adaptation of the mathematical and physical model through standard heuristics. The validation of temporal properties and behaviors do not scale up so well. Analysis of the difficulties suggests improvements in both tool support and formalism.

International audience; This paper presents our experience of modeling land transportation domain in the formal framework of Event-B. Well-specified requirements are crucial for good software design; they depend on the understanding of the domain. Thus, domain engineering becomes an essential activity. The possibility to have a formal model of a domain, consistent with the use of formal methods for developing critical software working within it, is an important issue. Safety-critical domains, like transportation, exhibit interesting features, such as high levels of non-determinism, complex interactions, stringent safety properties, multifaceted timing attributes, etc. The formal representation of these features is a challenging task. We explore the possibility of utilizing Event-B as a domain engineering tool. We discuss the problems we faced during this exercise and how we tackled them. Special attention is devoted to the issue of the validation of the model, in particular with a technique based on the animation of specifications. Event-B is mature enough to be an effective tool to model domains except in some areas, temporal properties mainly, where more work is still needed.

International audience; Well specified requirements are crucial for good software design and domain engineering helps better understanding and specification of requirements. Safety critical domains, such as transportation, exhibit interesting features, such as high levels of non-determinism, complex interactions, stringent safety properties, multifaceted timing attributes, etc. The formal representation of these features is a challenging task. This paper presents our experience of modeling land transportation domain in the formal framework of Event-B. We explore the possibility of using Event-B as a domain engineering tool. We discuss the problems posed by the introduction of time and how we tackle it. We design a technique based on animation to validate domain models.

Seeds of pea (Pisum sativum L.) were germinated for 5 days by soaking in distilled water or 5 mM cadmium chloride. Compared to the control, cadmium (Cd) caused a reduction in percent germination and embryo growth. Pyridine nucleotide coenzyme concentrations were determined in cotyledons and embryonic axis. Nicotinamide adenine dinucleotide (NADH) oxidase activity was examined. Cd treatment caused a restriction in levels of reduced coenzyme form in the mitochondria and the post-mitochondrial fraction of cotyledons, and embryonic axis. The oxidized coenzyme form has been accumulated by Cd-treated mitochondria of both tissues. It was also found that NADH oxidase activity was stimulated. The relationship between coenzyme levels, seed germination, pea growth, and Cd stress has been reported.

International audience; Glutaredoxins, proteins traditionally involved in redox reactions, are also required for iron–sulfur cluster assembly and haem biosynthesis. These new roles are probably related to the ability of some glutaredoxins to bind labile [2Fe–2S] clusters and to transfer them rapidly and efficiently to acceptor proteins. Recent results point to putative roles for glutaredoxins in the sensing of cellular iron and in iron–sulfur cluster biogenesis, either as scaffold proteins for the de novo synthesis of iron–sulfur clusters or as carrier proteins for the transfer of preformed iron–sulfur clusters. Based on prokaryote genome analysis and in vivo studies of iron regulation in yeast, we propose putative new roles and binding partners for glutaredoxins in the assembly of metalloproteins.

International audience; Photosynthesis occurs in vastly different forms, for e.g. some prokaryotes perform anoxygenic photosynthesis, and on the other hand, cyanobacteria, algae and land plants use oxygenic photosynthesis. Likewise, in land plants, most organisms rely on so-called C3 photosynthesis, but several tropical species as maize or sugarcane use a variant called C4 photosynthesis in which the first photosynthetic product is malate, a 4 carbon compound, rather than phosphoglyceric acid the more classical 3 carbon compound. Another example of the variation of the photosynthetic mode is found in so-called CAM (crassulacean acid metabolism) plants where CO2 fixation takes place at night rather than during the light, enabling these plants to resist extreme climatic conditions.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Former Editor-in-Chief and co-authors. The article includes content plagiarised from articles that have already appeared in the following journals. See: https://doi.org/10.1016/j.ecoenv.2010.05.015; International audience; Pea seeds (Pisum sativum L.) were germinated by soaking in H2O OR 5mM CdCl2 during a 5-day period. Enzyme activities involved in respiratory metabolism were studied in cotyledons. Mitochondrial cytochrome c oxidase and NADH- and succinate-cytochrome c reductase activities wer inhibited by cadmium treatment. The effects of Cd were performed in vivo and in vitro allowing to distinguish between the direct inhibition of the enzyme activities and the influence of the same enzymes into the cell environment. However, Cd exposure stimulated an enzyme activity of fermentation and inhibited the capacity of the enzyme inactivator (alcool dehydogenase inactivator). Moreover, the enzyme activities of NAD(P)H-recycling dehydogenases via secondary pentose phosphate pahtway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases, were enhanced in Cd-stressed seeds. The findings will help clarify the overall mechanisms that underlie cadmium-mediated toxicity in germinating seeds.

International audience; The recent release of several basidiomycete genome sequences allows an improvement of the classification of fungal glutathione S-transferases (GSTs). GSTs are well-known detoxification enzymes which can catalyze the conjugation of glutathione to non-polar compounds that contain an electrophilic carbon, nitrogen, or sulfur atom. Following this mechanism, they are able to metabolize drugs, pesticides, and many other xenobiotics and peroxides. A genomic and phylogenetic analysis of GST classes in various sequenced fungi—zygomycetes, ascomycetes, and basidiomycetes—revealed some particularities in GST distribution, in comparison with previous analyses with ascomycetes only. By focusing essentially on the wood-degrading basidiomycete Phanerochaete chrysosporium, this analysis highlighted a new fungal GST class named GTE, which is related to bacterial etherases, and two new subclasses of the omega class GSTs. Moreover, our phylogenetic analysis suggests a relationship between the saprophytic behavior of some fungi and the number and distribution of some GST isoforms within specific classes.

International audience; Thioredoxins (Trxs) are small oxidoreductases that are involved in redox homeostasis and are found in large numbers in the subcellular compartments of eukaryotic plant cells, including the chloroplasts. Also present in chloroplasts are two forms of thioredoxin reductase (TR), which use either NADPH or ferredoxin as an electron donor. In other compartments, two additional TR forms also use NADPH: one is distributed in all photosynthetic organisms and is similar to prokaryotic enzymes, whereas the other is restricted to algae and is similar to mammalian selenoproteins. Here, we review current knowledge of the different forms of TRs across organisms and discuss the possible evolutionary fate of this class of enzymes, which provide an example of convergent functional evolution.

International audience; Effect of heat treatment on extracellular enzymes involved in wood degradation by Trametes versicolor was investigated. Heat-treated and untreated beech blocks were exposed to T. versicolor on malt-agar medium and extracellular enzymatic activities investigated. A strong ABTS oxidizing activity has been detected during the first stage of colonization in both cases, while cellulase activities are mainly detected in the case of untreated beech wood. Further investigations carried out on holocellulose, isolated using sodium chlorite delignification procedure and subjected to heat treatment or not, indicate that commercially available cellulases and xylanases are able to hydrolyse untreated holocellulose, while heat-treated holocellulose was not affected. All these data suggest that chemical modifications of wood components during heat treatment disturb enzymatic system involved in wood degradation.

International audience; 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.

International audience; The initial stages of wood colonization and degradation by the white-rot Trametes versicolor have been investigated. T. versicolor was grown on Fagus sylvatica wood chips under solid-state fermentation in the presence of malt agar. The beginning of the colonization process was not associated with peroxidases production or hydrolytic activity. In contrast, a sharp induction of laccase expression was observed during the first colonization days. This induction is not due solely to fungal growth on wood chips since similar data were obtained in the presence of biocide (propiconazole)-treated wood, suggesting that enzyme production is due at least in part to the mycelium surrounding wood chips using nutrients from malt agar medium. Laccase production correlated with wood acetone extractives degradation, in particular with the oxidation of catechin, a major compound of beech extracts. In complementary experiments, woodextracted laccases were shown to be active with wood acetone extracts and pure catechin. Furthermore, wood extractives induced laccase expression. Taken together, these data suggest that the initial stages of wood colonization on malt agar by T. versicolor correlate with wood extractives degradation requiring laccase activity, whereas the other wood-degrading systems (peroxidases and polysaccharides hydrolases) are still repressed.

International audience; Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe–2S] cluster in two heterologously expressed chloro-plastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe–2S] cluster in apo-GrxS14. These Grxs possess the same mono-thiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe–S cluster assembly. In vitro kinetic studies monitored by CD spectro-scopy indicate that [2Fe–2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferred-oxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe–2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe–S clusters or in the regulation of the chloroplastic Fe–S cluster assembly machinery.

The cellular glutathione redox buffer is assumed to be part of signal transduction pathways transmitting environmental signals during biotic and abiotic stress, and thus is essential for regulation of metabolism and development. Ratiometric redox-sensitive GFP (roGFP) expressed in Arabidopsis thaliana reversibly responds to redox changes induced by incubation with H(2)O(2) or DTT. Kinetic analysis of these redox changes, combined with detailed characterization of roGFP2 in vitro, shows that roGFP2 expressed in the cytosol senses the redox potential of the cellular glutathione buffer via glutaredoxin (GRX) as a mediator of reversible electron flow between glutathione and roGFP2. The sensitivity of roGFP2 toward the glutathione redox potential was tested in vivo through manipulating the glutathione (GSH) content of wild-type plants, through expression of roGFP2 in the cytosol of low-GSH mutants and the endoplasmic reticulum (ER) of wild-type plants, as well as through wounding as an example for stress-induced redox changes. Provided the GSH concentration is known, roGFP2 facilitates the determination of the degree of oxidation of the GSH solution. Assuming sufficient glutathione reductase activity and non-limiting NADPH supply, the observed almost full reduction of roGFP2 in vivo suggests that a 2.5 mm cytosolic glutathione buffer would contain only 25 nm oxidized glutathione disulfide (GSSG). The high sensitivity of roGFP2 toward GSSG via GRX enables the use of roGFP2 for monitoring stress-induced redox changes in vivo in real time. The results with roGFP2 as an artificial GRX target further suggest that redox-triggered changes of biologic processes might be linked directly to the glutathione redox potential via GRX as the mediator.