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Thioredoxins are ubiquitous small-molecular-weight proteins (typically 100-120 amino-acid residues) containing an extremely reactive disulphide bridge with a highly conserved sequence -Cys-Gly(Ala/Pro)-Pro-Cys-. In bacteria and animal cells, thioredoxins participate in multiple reactions which require reduction of disulphide bonds on selected target proteins/ enzymes. There is now ample biochemical evidence that thioredoxins exert very specific functions in plants, the best documented being the redox regulation of chloroplast enzymes. Another area in which thioredoxins are believed to play a prominent role is in reserve protein mobilization during the process of germination. It has been discovered that thioredoxins constitute a large multigene family in plants with different-subcellular localizations, a unique feature in living cells so far. Evolutionary studies based on these molecules will be discussed, as well as the available biochemical and genetic evidence related to their functions in plant cells. Eukaryotic photosynthetic plant cells are also unique in that they possess two different reducing systems, one extrachloroplastic dependent on NADPH as an electron donor, and the other one chloroplastic, dependent on photoreduced ferredoxin. This review will examine in detail the latest progresses in the area of thioredoxin structural biology in plants, this protein being an excellent model for this purpose. The structural features of the reducing enzymes ferredoxin thioredoxin reductase and NADPH thioredoxin reductase will also be described. The properties of the target enzymes known so far in plants will be detailed with special emphasis on the structural features which make them redox regulatory. Based on sequence analysis, evidence will be presented that redox regulation of enzymes of the biosynthetic pathways first appeared in cyanobacteria possibly as a way to cope with the oxidants produced by oxygenic photosynthesis. It became more elaborate in the chloroplasts of higher plants where a co-ordinated functioning of the chloroplastic and extra chloroplastic metabolisms is required. CONTENTS Summary 543 I. Introduction 544 II. Thioredoxins from photosynthetic organisms as a structural model 545 III. Physiological functions 552 IV. The thioredoxin reduction systems 556 V. Structural aspects of target enzymes 558 VI. Concluding remarks 563 Acknowledgements 564 References 564.

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 fluorescence properties of FAD of Thermotoga maritima thioredoxin reductase (TmTR), taken together with the amino acid sequences and structures of similar TRs, are consistent with the interdomain rotation in the catalysis of TmTR. The standard redox potential of FAD of TmTR, –0.230 V, determined by the reactions with 3-acetylpyridine adenine dinucleotide (APAD+/APADH) redox couple, is close to that of E. coli TR. During the reduction of duroquinone with TmTR, the transient formation of neutral FAD semiquinone, and, possibly, FADH2–NAD+ complex was observed. This shows that in spite of obligatory twoelectron (hydride)-transfer between NADH and physiological disulfide oxidants, the FAD cofactor of TmTR may exist under a stable semiquinone form.

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.

Fungal degradation of wood is mainly restricted to basidiomycetes, these organisms having developed complex oxidative and hydrolytic enzymatic systems. Besides these systems, wood-decaying fungi possess intracellular networks allowing them to deal with the myriad of potential toxic compounds resulting at least in part from wood degradation but also more generally from recalcitrant organic matter degradation. The members of the detoxification pathways constitute the xenome. Generally, they belong to multigenic families such as the cytochrome P450 monooxygenases and the glutathione transferases. Taking advantage of the recent release of numerous genomes of basidiomycetes, we show here that these multigenic families are extended and functionally related in wood-decaying fungi. Furthermore, we postulate that these rapidly evolving multigenic families could reflect the adaptation of these fungi to the diversity of their substrate and provide keys to understand their ecology. This is of particular importance for white biotechnology, this xenome being a putative target for improving degradation properties of these fungi in biomass valorization purposes.

Thermotoga maritima peroxiredoxin-nitroreductase hybrid enzyme (Prx-NR) consists of a FMN-containing nitroreductase (NR) domain fused to a peroxiredoxin (Prx) domain. These domains seem to function independently as no electron transfer occurs between them. The reduction of quinones and nitroaromatics by NR proceeded in a two-electron manner, and follows a 'ping-pong' scheme with sometimes pronounced inhibition by quinone substrate. The comparison of steady- and presteady-state kinetic data shows that in most cases, the oxidative half-reaction may be rate-limiting in the catalytic cycle of NR. The enzyme was inhibited by dicumarol, a classical inhibitor of oxygen-insensitive nitroreductases. The reduction of quinones and nitroaromatic compounds by Prx-NR was characterized by the linear dependence of their reactivity (logk(cat)/K(m)) on their single-electron reduction potentials E(7)(1), while the reactivity of quinones markedly exceeded the one with nitroaromatics. It shows that NR lacks the specificity for the particular structure of these oxidants, except their single-electron accepting potency and the rate of electron self-exchange. It points to the possibility of a single-electron transfer step in a net two-electron reduction of quinones and nitroaromatics by T. maritima Prx-NR, and to a significant diversity of the structures of flavoenzymes which may perform the two-electron reduction of quinones and nitroaromatics.

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.

Legumes interact symbiotically with bacteria of the Rhizobiaceae to form nitrogen-fixing root nodules. We investigated the contribution of the three glutaredoxin (Grx)-encoding genes present in the Sinorhizobium meliloti genome to this symbiosis. SmGRX1 (CGYC active site) and SmGRX3 (CPYG) recombinant proteins displayed deglutathionylation activity in the 2-hydroethyldisulfide assay, whereas SmGRX2 (CGFS) did not. Mutation of SmGRX3 did not affect S. meliloti growth or symbiotic capacities. In contrast, SmGRX1 and SmGRX2 mutations decreased the growth of free-living bacteria and the nitrogen fixation capacity of bacteroids. Mutation of SmGRX1 led to nodule abortion and an absence of bacteroid differentiation, whereas SmGRX2 mutation decreased nodule development without modifying bacteroid development. The higher sensitivity of the Smgrx1 mutant strain as compared with wild-type strain to oxidative stress was associated with larger amounts of glutathionylated proteins. The Smgrx2 mutant strain displayed significantly lower levels of activity than the wild type for two iron-sulfur-containing enzymes, aconitase and succinate dehydrogenase. This lower level of activity could be associated with deregulation of the transcriptional activity of the RirA iron regulator and higher intracellular iron content. Thus, two S. meliloti Grx proteins are essential for symbiotic nitrogen fixation, playing independent roles in bacterial differentiation and the regulation of iron metabolism.