The conformational stability and biophysical properties of the eukaryotic thioredoxins of Pisum sativum are not family-conserved

Thioredoxins (TRXs) are ubiquitous proteins involved in redox processes. About forty genes encode TRX or TRX-related proteins in plants, grouped in different families according to their subcellular localization. For instance, the h-type TRXs are located in cytoplasm or mitochondria, whereas f-type TRXs have a plastidial origin, although both types of proteins have an eukaryotic origin as opposed to other TRXs. Herein, we study the conformational and the biophysical features of TRXh1, TRXh2 and TRXf from Pisum sativum. The modelled structures of the three proteins show the well-known TRX fold. While sharing similar pH-denaturations features, the chemical and thermal stabilities are different, being PsTRXh1 (Pisum sativum thioredoxin h1) the most stable isoform; moreover, the three proteins follow a three-state denaturation model, during the chemical-denaturations. These differences in the thermal- and chemical-denaturations result from changes, in a broad sense, of the several ASAs (accessible surface areas) of the proteins. Thus, although a strong relationship can be found between the primary amino acid sequence and the structure among TRXs, that between the residue sequence and the conformational stability and biophysical properties is not. We discuss how these differences in the biophysical properties of TRXs determine their unique functions in pea, and we show how residues involved in the biophysical features described (pH-titrations, dimerizations and chemical-denaturations) belong to regions involved in interaction with other proteins. Our results suggest that the sequence demands of protein-protein function are relatively rigid, with different protein-binding pockets (some in common) for each of the three proteins, but the demands of structure and conformational stability per se (as long as there is a maintained core), are less so.     

Thioredoxins m are major players in the multifaceted light-adaptive response in Arabidopsis thaliana

Thioredoxins (TRXs) are well-known redox signalling players, which carry out post-translational modifications in target proteins. Chloroplast TRXs are divided into different types and have central roles in light energy uptake and the regulation of primary metabolism. The isoforms TRX m1, m2, and m4 from Arabidopsis thaliana are considered functionally related. Knowing their key position in the hub of plant metabolism, we hypothesized that the impairment of the TRX m signalling would not only have harmful consequences on chloroplast metabolism but also at different levels of plant development. To uncover the physiological and developmental processes that depend on TRX m signalling, we carried out a comprehensive study of Arabidopsis single, double, and triple mutants defective in the TRX m1, m2, and m4 proteins. As light and redox signalling are closely linked, we investigated the response to high light (HL) of the plants that are gradually compromised in TRX m signalling. We provide experimental evidence relating the lack of TRX m and the appearance of novel phenotypic features concerning mesophyll structure, stomata biogenesis, and stomatal conductance. We also report new data indicating that the isoforms of TRX m fine-tune the response to HL, including the accumulation of the protective pigment anthocyanin. These results reveal novel signalling functions for the TRX m and underline their importance for plant growth and fulfilment of the acclimation/response to HL conditions.     

Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis

Thioredoxins (TRXs) mediate light‐dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox‐regulated enzymes. Of the two plastid TRX systems, the ferredoxin‐TRX system consists of ferredoxin‐thioredoxin reductase (FTR) and multiple TRXs, while the NADPH‐dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd‐TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO₂ fixation rate and lowered non‐photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX‐regulated enzymes in Calvin–Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose‐1,6‐bisphosphatase, phosphoribulokinase and CF₁γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC‐mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin‐TRX system and is crucial when availability of light is limiting photosynthesis.     

Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7-A

The structure of a thaumatin-like protein from banana (Musa acuminata) fruit, an allergen with antifungal properties, was solved at 1.7-A-resolution, by X-ray crystallography. Though the banana protein exhibits a very similar overall fold as thaumatin it markedly differs from the sweet-tasting protein by the presence of a surface exposed electronegative cleft. Due to the presence of this electronegative cleft, the banana thaumatin-like protein (Ban-TLP) acquires a strong (local) electronegative character that eventually explains the observed antifungal activity. Our structural analysis also revealed the presence of conserved residues of exposed epitopic determinants that are presumably responsible for the allergenic properties of banana fruit towards susceptible individuals, and provided evidence that the Ban-TLP shares some structurally highly conserved IgE-binding epitopes with thaumatin-like proteins from fruits or pollen from other plants. In addition, some overlap was detected between the predicted IgE-binding epitopes of the Ban-TLP and IgE-binding epitopes previously identified in the mountain cedar Jun a 3 TLP aeroallergen. The presence of these common epitopes offers a molecular basis for the cross-reactivity between aeroallergens and fruit allergens.     

Molecular cloning and characterization of a thioredoxin gene from Echinococcus granulosus.

The insert of a clone from a lambdagt11 Echinococcus granulosus (Platyhelminth, Cestoda) protoscolex cDNA library, showed an open reading frame whose deduced protein sequence presents a high homology with all described thioredoxins (TRX). The TRX active site (Trp-Cys-Gly-Pro-Cys) is completely conserved. With a monospecific antibody, selected from a total anti-protoscolex sera by the isolated clone, a 12 kDa polypeptide was immunoprecipitated from a protoscolex total protein extract. Furthermore, an antiserum raised against a recombinant EgTRX also recognizes a 12 kDa band in these extracts. The recombinant protein presents TRX activity, using the insulin reduction assay. Finally, a TRX activity was characterized in protoscolex extracts. In all organisms where TRXs were studied, they participate in a cascade of redox exchanges, contributing to the maintaining of cell homeostasis. Considering that the parasitic flatworm E. granulosus is probably submitted to an important oxidative stress due to host defences, EgTRX protein could be involved in the survival strategies of this parasite.     

Immunocytochemical localization of Pisum sativum TRXs f and m in non-photosynthetic tissues

Plants are the organisms containing the most complex multigenic family for thioredoxins (TRX). Several types of TRXs are targeted to chloroplasts, which have been classified into four subgroups: m, f, x, and y. Among them, TRXs f and m were the first plastidial TRXs characterized, and their function as redox modulators of enzymes involved in carbon assimilation in the chloroplast has been well-established. Both TRXs, f and m, were named according to their ability to reduce plastidial fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (MDH), respectively. Evidence is presented here based on the immunocytochemistry of the localization of f and m-type TRXs from Pisum sativum in non-photosynthetic tissues. Both TRXs showed a different spatial pattern. Whilst PsTRXm was localized to vascular tissues of all the organs analysed (leaves, stems, and roots), PsTRXf was localized to more specific cells next to xylem vessels and vascular cambium. Heterologous complementation analysis of the yeast mutant EMY63, deficient in both yeast TRXs, by the pea plastidial TRXs suggests that PsTRXm, but not PsTRXf, is involved in the mechanism of reactive oxygen species (ROS) detoxification. In agreement with this function, the PsTRXm gene was induced in roots of pea plants in response to hydrogen peroxide.     

Heterologous complementation of yeast reveals a new putative function for chloroplast m-type thioredoxin

In the chloroplast of higher plants, two types of thioredoxins (TRX), namely TRX m which shows high similarity to prokaryotic thioredoxins and TRX f which is more closely related to eukaryotic thioredoxins, have been found and biochemically characterized, but little is known about their physiological specificity with respect to their target(s). Here, we tested, in vivo, the ability of organelle-specific TRX from Arabidopsis thaliana to compensate for TRX deficiency of a Saccharomyces cerevisiae mutant strain. Seven plant organellar TRX (four of the m type, two of the f type and a newly discovered TRX x of prokaryotic type) were expressed in yeast in a putative mature form. None of these heterologous TRX were able to restore growth on sulphate or methionine sulphoxide of the mutant cells. When we tested their ability to rescue the oxidant-hypersensitive phenotype of the TRX-deficient strain, we found that TRX m and TRX x, but not TRX f, affected the tolerance to oxidative stress induced by either hydrogen peroxide or an alkyl hydroperoxide. Athm1, Athm2, Athm4 and Athx induced hydrogen peroxide tolerance like the endogenous yeast thioredoxins. Unexpectedly, Athm3 had a hypersensitizing effect towards oxidative stress. The presence of functional heterologous TRX was checked in the recombinant clones tested, supporting distinct abilities for organelle-specific plant TRX to compensate for TRX deficiency in yeast. We propose a new function for the prokaryotic-type chloroplastic TRX as an anti-oxidant and provide in vivo evidence for different roles of chloroplastic TRX isoforms.     

Recognition between a short unstructured peptide and a partially folded fragment leads to the thioredoxin fold sharing native-like dynamics

Thioredoxins (TRXs) constitute attractive α/β scaffolds for investigating molecular recognition. The interaction between the recombinant fragment spanning the sequence 1-93 of full-length TRX (TRX1-93) and the synthetic peptide comprising residues 94-108 (TRX94-108), plus a C-terminal tyrosine tag (the numbering scheme used in entry pdb 2TRX is used throughout the article, two complementary moieties of E. coli TRX, brings about the consolidation of a native-like complex. Despite its reduced thermodynamic stability, this complex is able to acquire fine structural features remarkably similar to those characteristic of full-length TRX, namely, hydrodynamic behavior, assessed by diffusion-ordered spectroscopy (DOSY)-NMR; the pattern of secondary structure, as revealed by three-bond HNHα coupling constants and secondary shifts for Hα/CO/Cα/Cβ; native-like tertiary structural signatures revealed by near-UV circular dichroism (CD) spectroscopy. The complex exhibits a relaxation behavior compatible with that expected for a native-like structure. However, heteronuclear nuclear Overhauser effect (NOE)s reveal an enhanced dynamics for the complex by comparison with full-length TRX. Furthermore, higher R(2) values for residues 43-50 and 74-89 would likely result from an exchange process modulated by the peptide at the interface region. The slow kinetics of the consolidation reaction was followed by CD and real-time NMR. Equilibrium titration experiments by NMR yield a K(D) value of 1.4 ± 1.0 μM and a second low-affinity (>150 μM) binding event in the vicinity of the active site. Molecular dynamics simulations of both the isolated fragment TRX1-93 and the complex suggest the destabilization of α2 and α3 helical elements and the persistence of β-structure in the absence of TRX94-108. Altogether, structural and dynamic evidence presented herein points to the key role played by the C-terminal helix in establishing the overall fold. This critical switch module endows reduced TRX with the ability to act as a cooperative folding unit.     

NADP-malate dehydrogenase from unicellular green alga Chlamydomonas reinhardtii. A first step toward redox regulation?

The determinants of the thioredoxin (TRX)-dependent redox regulation of the chloroplastic NADP-malate dehydrogenase (NADP-MDH) from the eukaryotic green alga Chlamydomonas reinhardtii have been investigated using site-directed mutagenesis. The results indicate that a single C-terminal disulfide is responsible for this regulation. The redox midpoint potential of this disulfide is less negative than that of the higher plant enzyme. The regulation is of an all-or-nothing type, lacking the fine-tuning provided by the second N-terminal disulfide found only in NADP-MDH from higher plants. The decreased stability of specific cysteine/alanine mutants is consistent with the presence of a structural disulfide formed by two cysteine residues that are not involved in regulation of activity. Measurements of the ability of C. reinhardtii thioredoxin f (TRX f) to activate wild-type and site-directed mutants of sorghum (Sorghum vulgare) NADP-MDH suggest that the algal TRX f has a redox midpoint potential that is less negative than most those of higher plant TRXs f. These results are discussed from an evolutionary point of view.     

The Glutaredoxin Family in Oxygenic Photosynthetic Organisms

Glutaredoxins (GRXs) are small redox proteins of the thioredoxin (TRX) superfamily. Compared to TRXs, much less information on the GRX family is available, especially in photosynthetic organisms since GRXs have been mainly studied in E. coli, yeast and mammal cells. The analysis of the TRX family in oxygenic photosynthetic organisms revealed an unsuspected multiplicity of TRXs but it is not known if the same situation holds for GRXs. Despite the availability of genome sequences from different oxygenic photosynthetic organisms, the number of GRXs and the different groups present in these organisms are still undescribed. This paper presents a comparative analysis of the GRX families present in Arabidopsis, Chlamydomonas and Synechocystis which were found to contain 30, 6 and 3 GRX genes, respectively. The putative subcellular localization of each GRX and its relative expression level, based on EST data, have been investigated. This analysis reveals the presence of three major classes of GRXs, the CPYC type, the CGFS type and a previously undescribed type, called the CC type that appears specific to higher plants. These data are discussed in view of recent results suggesting a complex cross-regulation between the TRX and GRX systems.     

Genome- Wide Analysis and Characterization of the TRX Gene Family in Upland Cotton

Thioredoxins (TRX) are small molecules of proteins that are present in all organisms. TRXs play an important role in diverse functions of plant growth and development. In this study, we performed genome-wide, characterization and expression levels of TRX gene family in cotton. A total of 150 GhTRX proteins were identified in upland cotton and classified into five subfamilies based on their domain compositions. Phylogenetic tree analysis divided TRX genes into seven subgroups. GhTRX genes covered all upland cotton chromosomes, with duplicated gene events. Ka/Ks ratio of three gene pairs was less than 1, suggesting purifying selection. The functions of GhTRX genes were studied using gene ontology, protein localization, and promoter analysis. Furthermore, six GhTRX genes were randomly selected to examine their expression level in cotton development and under various exogenous treatments. The genes showed high expressions in various tissues and at different stages of leaf senescence, also showed high expression under abscisic acid, ethylene, drought, and salinity. This study reveals the first report of TRX family genes in upland cotton. However further studies are needed to elucidate their specific functions in cotton plant.     

Heavy-Metal Regulation of Thioredoxin Gene Expression in Chlamydomonas reinhardtii

Heavy metals are highly toxic compounds for cells. In this report we demonstrate that the expression of Chlamydomonas reinhardtii thioredoxins (TRX) m and h is induced by heavy metals. Upon exposure of the cells to Cd and Hg, a strong accumulation of both messengers was observed. Western-blot experiments revealed that among these two TRXs, only TRX h polypeptides accumulated in response to the toxic cations. A biochemical analysis indicated that heavy metals inhibit TRX activity, presumably by binding at the level of their active site. Sequence analysis of the C. reinhardtii TRX h promoter revealed the presence of cis-acting elements related to cadmium induction. The origins and purposes of this regulation are discussed. Our data suggest, for the first time to our knowledge, a possible implication of TRXs in defense mechanisms against heavy metals.     

The thioredoxin superfamily in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The thioredoxin (TRX) superfamily includes redox proteins such as thioredoxins, glutaredoxins (GRXs) and protein disulfide isomerases (PDI). These proteins share a common structural motif named the thioredoxin fold. They are involved in disulfide oxido-reduction and/or isomerization. The sequencing of the Arabidopsisgenome revealed an unsuspected multiplicity of TRX and GRX genes compared to other organisms. The availability of full Chlamydomonasgenome sequence offers the opportunity to determine whether this multiplicity is specific to higher plant species or common to all photosynthetic eukaryotes. We have previously shown that the multiplicity is more limited in Chlamydomonas for TRX and GRX families. We extend here our analysis to the PDI family. This paper presents a comparative analysis of the TRX, GRX and PDI families present in Arabidopsis,Chlamydomonas and Synechocystis. The putative subcellular localization of each protein and its relative expression level, based on EST data, have been investigated. This analysis provides a large overview of the redox regulatory systems present in Chlamydomonas. The data are discussed in view of recent results suggesting a complex cross-talk between the TRX, GRX and PDI redox regulatory networks.     

Redox regulation of chloroplast metabolism

Regulation of enzyme activity based on thiol-disulfide exchange is a regulatory mechanism in which the protein disulfide reductase activity of thioredoxins (TRXs) plays a central role. Plant chloroplasts are equipped with a complex set of up to 20 TRXs and TRX-like proteins, the activity of which is supported by reducing power provided by photosynthetically reduced ferredoxin (FDX) with the participation of a FDX-dependent TRX reductase (FTR). Therefore, the FDX–FTR–TRXs pathway allows the regulation of redox-sensitive chloroplast enzymes in response to light. In addition, chloroplasts contain an NADPH-dependent redox system, termed NTRC, which allows the use of NADPH in the redox network of these organelles. Genetic approaches using mutants of Arabidopsis (Arabidopsis thaliana) in combination with biochemical and physiological studies have shown that both redox systems, NTRC and FDX-FTR-TRXs, participate in fine-tuning chloroplast performance in response to changes in light intensity. Moreover, these studies revealed the participation of 2-Cys peroxiredoxin (2-Cys PRX), a thiol-dependent peroxidase, in the control of the reducing activity of chloroplast TRXs as well as in the rapid oxidation of stromal enzymes upon darkness. In this review, we provide an update on recent findings regarding the redox regulatory network of plant chloroplasts, focusing on the functional relationship of 2-Cys PRXs with NTRC and the FDX–FTR–TRXs redox systems for fine-tuning chloroplast performance in response to changes in light intensity and darkness. Finally, we consider redox regulation as an additional layer of control of the signaling function of the chloroplast.

Thiol-dependent redox regulatory and antioxidant systems act concertedly to modulate chloroplast metabolism and signaling function.

Advances

• Plant chloroplasts harbor a complex redox network composed of the FDX–FTR–TRXs pathway, linking redox regulation to light, and NTRC, an NADPH-dependent system required for the activity of TRXs. Both systems adjust chloroplast performance to environmental cues.
• A relevant function of NTRC is redox control of 2-Cys PRXs, which maintains the reductive activity of chloroplast TRXs in the light. The NTRC–2-Cys PRXs redox system helps fine-tune the redox state of chloroplast enzymes thereby adjusting photosynthetic performance to changes in light.
• 2-Cys PRXs participate in the rapid oxidative inactivation of chloroplast enzymes in the dark, mediating the transfer of reducing equivalents from reduced enzymes, via TRXs, to hydrogen peroxide.
• Involvement of redox regulation in chloroplast retrograde signaling modulates early stages of plant development and response to environmental stress.

     

PsTRXh1 and PsTRXh2 are both pea h-type thioredoxins with antagonistic behavior in redox imbalances

Thioredoxins (TRXs) are small ubiquitous oxidoreductases involved in disulfide bond reduction of a large panel of target proteins. The most complex cluster in the family of plant TRXs is formed by h-type TRXs. In Arabidopsis (Arabidopsis thaliana), nine members of this subgroup were described, which are less well known than their plastidial counterparts. The functional study of type-h TRXs is difficult because of the high number of isoforms and their similar biochemical characteristics, thus raising the question whether they have specific or redundant functions. Type-h TRXs are involved in seed germination and self incompatibility in pollen-pistil interaction. Their function as antioxidants has recently been proposed, but further work is needed to clarify this function in plants. In this study, we describe two new h-type TRXs from pea (Pisum sativum; stated PsTRXh1 and PsTRXh2). By functional complementation of a yeast (Saccharomyces cerevisiae) trx1Delta trx2Delta double mutant, we demonstrate that PsTRXh1 is involved in the redox-imbalance control, possibly through its interaction with peroxiredoxins. In contrast, PsTRXh2 provokes a phenotype of hypersensitivity to hydrogen peroxide in the yeast mutant. Furthermore, we show differential gene expression and protein accumulation of the two isoforms, PsTRXh1 protein being abundantly detected in vascular tissue and flowers, whereas PsTRXh2 gene expression was hardly detectable. By comparison with previous data of additional PsTRXh isoforms, our results indicate specific functions for the pea h-type TRXs so far described.