The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

Intron position as an evolutionary marker of thioredoxins and thioredoxin domains

In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants. All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic. We have cloned and sequencedArabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h. In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron. The recently published sequence ofChlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants. This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae. In addition, we have cloned and sequenced pea DNA genomic fragments encoding thioredoxins m and f. The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this sequence and suggesting that its association with the transit peptide has been facilitated by exon shuffling. In contrast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomerases. This strongly supports the hypothesis of a eukaryotic origin for chloroplastic thioredoxin f.     

NMR structures of thioredoxin m from the green alga Chlamydomonas reinhardtii

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.     

Chlamydomonas reinhardtii thioredoxins: structure of the genes coding for the chloroplastic m and cytosolic h isoforms; expression in Escherichia coli of the recombinant proteins,purification and biochemical properties

Based on known amino acid sequences, probes have been generated by PCR and used for the subsequent isolation of cDNAs and genes coding for two thioredoxins (m and h) of Chlamydomonas reinhardtii. Thioredoxin m, a chloroplastic protein, is encoded as a preprotein of 140 amino acids (15 101 Da) containing a transit peptide of 34 amino acids with a very high content of Ala and Arg residues. The sequence for thioredoxin h codes for a 113 amino acid protein with a molecular mass of 11817 Da and no signal sequence. The thioredoxin m gene contains a single intron and seems to be more archaic in structure than the thioredoxin h gene, which is split into 4 exons. The cDNA sequences encoding C. reinhardtii thioredoxins m and h have been integrated into the pET-3d expression vector, which permits efficient production of proteins in Escherichia coli cells. A high expression level of recombinant thioredoxins was obtained (up to 50 mg/l culture). This has allowed us to study the biochemical/biophysical properties of the two recombinant proteins. Interestingly, while the m-type thioredoxin was found to have characteristics very close to the ones of prokaryotic thioredoxins, the h-type thioredoxin was quite different with respect to its kinetic behaviour and, most strikingly, its heat denaturation properties.Abbreviations DTT dithiothreitol - FBPase Fructose 1,6-biphosphate phosphatase - FTR ferredoxin-thioredoxin reductase - IPTG isopropyl thiogalactoside - NADP-MDH NADPH-dependent malate dehydrogenase - NMR nuclear magnetic resonance - NTR NADPH-dependent thioredoxin reductase Dedicated to the memory of Claude Crétin     

Cytosolic, Mitochondrial Thioredoxins and Thioredoxin Reductases in Arabidopsis Thaliana

Thioredoxins, by reducing disulfide bridges are one of the main participants that regulate cellular redox balance. In plants, the thioredoxin system is particularly complex. The most well-known thioredoxins are the chloroplastic ones, that participate in the regulation of enzymatic activities during the transition between light and dark phases. The mitochondrial system composed of NADPH-dependent thioredoxin reductase and type o thioredoxin has only recently been described. The type h thioredoxin group is better known. Yeast complementation experiments demonstrated that Arabidopsis thaliana thioredoxins h have divergent functions, at least in Saccharomyces cerevisiae. They have diverse affinities for different target proteins, most probably because of structural differences. However, plant thioredoxin h functions still have to be defined.     

Evolution of redoxin genes in the green lineage

The availability of the Arabidopsis genome revealed the complexity of the gene families implicated in dithiol disulfide exchanges. Most non-green organisms present less dithiol oxidoreductase genes. The availability of the almost complete genome sequence of rice now allows a systematic search for thioredoxins, glutaredoxins and their reducers. This shows that all redoxin families previously defined for Arabidopsis have members in the rice genome and that all the deduced rice redoxins fall within these families. This establishes that the redoxin classification applies both to dicots and monocots. Nevertheless, within each redoxin type the number of members is not the same in these two higher plants and it is not always possible to define orthologues between rice and Arabidopsis. The sequencing of two unicellular algae (Chlamydomonas and Ostreococcus) genomes are almost finished. This allowed us to follow the origin of the different gene families in the green lineage. It appears that most thioredoxin and glutaredoxin types, their chloroplastic, mitochondrial and cytosolic reducers are always present in these unicellular organisms. Nevertheless, striking differences appear in comparison to higher plant redoxins. Some thioredoxin types are not present in these algal genomes including thioredoxins o, clot and glutaredoxins CCxC. Numerous redoxins, including the cytosolic thioredoxins, do not fit with the corresponding higher plant classification. In addition both algae present a NADPH-dependent thioredoxin reductase with a selenocysteine which is highly similar to the animal thioredoxin reductases, a type of thioredoxin reductase not present in higher plants. An erratum to this article can be found at     

Redox control of hydrogenase activity in the green alga Scenedesmus obliquus by thioredoxin and other thiols.

The activity of the NiFe-hydrogenase from the green alga Scenedesmus obliquus is inhibited by both algal thioredoxins f and I+II, and by Escherichia coli thioredoxin. The strongest inhibition was observed with homologous chloroplastic thioredoxin f (I50 = 21 nM) and E. coli thioredoxin (I50 = 83 nM). For the homologous cytoplasmic thioredoxins I+II an I50 of 667 nM was determined. Glutathione shows a similar but much less pronounced inhibitory effect whereas dithiothreitol had no effect. In addition to glucose-6-phosphate dehydrogenase, NiFe-hydrogenase is only the second enzyme known to be inhibited by reduced thioredoxin.     

Contrasting evolutionary histories of chloroplast thioredoxins f and m

Fourteen thioredoxin sequences were used to construct a minimal phylogenetic tree by using parsimony. The bacterial thioredoxins clustered into three groups: one containing the photosynthetic purple bacteria, Escherichia and Corynebacterium; a second containing the photosynthetic green bacterium, Chlorobium; and a third containing cyanobacteria. These groupings are similar to those generated from earlier 16s RNA analyses. Animal thioredoxins formed a fourth group. The two thioredoxins of chloroplasts (f and m) showed contrasting phylogenetic patterns. As predicted from prior studies, spinach chloroplast thioredoxin m grouped with its counterparts from cyanobacteria and eukaryotic algae, but, unexpectedly, thioredoxin f grouped with the animal thioredoxins. The results indicate that, during evolution, thioredoxin m of contemporary photosynthetic eukaryotic cells was derived from a prokaryotic symbiont, whereas thioredoxin f descended from an ancestral eukaryote common to plants and animals. The findings illustrate the potential of thioredoxin as a phylogenetic marker and suggest a relationship between the animal and f-type thioredoxins.      

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.     

The Arabidopsis plastidial thioredoxins: new functions and new insights into specificity

The sequencing of the genome of Arabidopsis thaliana revealed that this plant contained numerous isoforms of thioredoxin (Trx), a protein involved in thiol-disulfide exchanges. On the basis of sequence comparison, seven putative chloroplastic Trxs have been identified, four belonging to the m-type, two belonging to the f-type, and one belonging to a new x-type. In the present work, these isoforms were produced and purified as recombinant proteins without their putative transit peptides. Their activities were tested with two known chloroplast thioredoxin targets: NADP-malate dehydrogenase and fructose-1,6-bisphosphatase and also with a chloroplastic 2-Cys peroxiredoxin. The study confirms the strict specificity of fructose-bisphosphatase for Trx f, reveals that some Trxs are unable to activate NADP-malate dehydrogenase, and shows that the new x-type is the most efficient substrate for peroxiredoxin while being inactive toward the two other targets. This suggests that this isoform might be specifically involved in resistance against oxidative stress. Three-dimensional modeling shows that one of the m-type Trxs, Trx m3, which has no activity with any of the three targets, exhibits a negatively charged surface surrounding the active site. A green fluorescent protein approach confirms the plastidial localization of these Trxs.     

Poplar peroxiredoxin Q. A thioredoxin-linked chloroplast antioxidant functional in pathogen defense

Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.     

Interactions between nitrogen oxide-containing compounds and peripheral benzodiazepine receptors.

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii have been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic -helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C-terminal region with the potential to form an amphiphilic β-strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N-tenninal domain similar to stroma-targeting transit peptides attached to a C-terminal apolar domain that share many characteristics with secretory signal peptides.     

Isolation and characterization of different forms of thioredoxins from the green alga Acetabularia mediterranea: identification of an NADP/thioredoxin system in the extrachloroplastic fraction.

A procedure has been developed for the simultaneous purification to apparent homogeneity of chloroplast thioredoxins f and m, and nonchloroplast thioredoxin h, from the green alga Acetabularia mediterranea. In the chloroplast fraction, three thioredoxins were isolated: one f type thioredoxin (Mr 13.4 kDa) and two m type thioredoxin forms (Mr of 12.9 and 13.8 kDa). A Western blot analysis of crude and purified chloroplast thioredoxin preparations revealed that Acetabularia thioredoxin m was immunologically related to its higher-plant counterparts whereas thioredoxin f was not. In the nonchloroplast fraction, a single form of thioredoxin h (Mr 13.4 kDa) and its associated enzyme NADP-thioredoxin reductase (NTR) were evidenced. Acetabularia NTR was partially purified and shown to be an holoenzyme composed of two 33.0-kDa subunits as is the case for other plant and bacterial NTRs. Similarity was confirmed by immunological tests: the algal enzyme was recognized by antibodies to spinach and Escherichia coli NTRs. Acetabularia thioredoxin h seemed to be more distant from higher-plant type h thioredoxins as recognition by antibodies to thioredoxin h from spinach and wheat was weak. The algal thioredoxin h was also slightly active with spinach and E. coli NTRs. These results suggest that in green algae as in the green tissues of higher plants the NADP and chloroplast thioredoxin systems are present simultaneously, and might play an important regulatory role in their respective cellular compartments.     

Comments on ''Reaction mechanism of flavin-dependent hydroxylation: evolution of a non-imitable enzyme'' by C. M. Visser. Research note on the possible occurrence of dioxetanes and other cyclic peroxides in flavin and pteridine-mediated hydroxylations

A new method of purification of chloroplastic thioredoxins has been presented. This method is based on affinity chromatography on fructose-bisphosphatase--Sepharose columns. Two thioredoxin, fA and fB, may be extracted and purified to homogeneity from the same leaf extract. Whereas fA is monomeric and has an Mr of 11 400 +/- 500, fB is dimeric with an Mr of 18 000 +/- 600. The dimer dissociates in two halves in the ultracentrifuge under the effect of high pressures. Raising the ionic strength results in the same effect. Thioredoxins fA and fB activate to similar extents chloroplastic fructose bisphosphatase and NADP--malate dehydrogenase. Chloroplastic sedoheptulose bisphosphatase is activated by thioredoxin fB but not by thioredoxin fA.     

Involvement of CDSP 32, a drought-induced thioredoxin, in the response to oxidative stress in potato plants

In animal cells, yeast and bacteria, thioredoxins are known to participate in the response to oxidative stress. We recently identified a novel type of plant thioredoxin named CDSP 32 for chloroplastic drought-induced stress protein of 32 kDa. In the present work, we measured comparable increases in the glutathione oxidation ratio and in the level of chlorophyll thermoluminescence, a specific marker for thylakoid lipid peroxidation in Solanum tuberosum plants subjected to drought or oxidative treatments (photooxidative stress, gamma irradiation and methyl viologen spraying). Further, substantial accumulations of CDSP 32 mRNA and protein were revealed upon oxidative treatments. These data show for the first time in plants the induction of a thioredoxin by oxidative stress. We conclude that CDSP 32 may preserve chloroplastic structures against oxidative injury upon drought.     

Purification, characterization, and complete amino acid sequence of a thioredoxin from a green alga, Chlamydomonas reinhardtii

Two thioredoxins (named Ch1 and Ch2 in reference to their elution pattern on an anion-exchange column) have been purified to homogeneity from the green alga, Chlamydomonas reinhardtii. In this paper, we described the properties and the sequence of the most abundant form, Ch2. Its activity in various enzymatic assays has been compared with those of Escherichia coli and spinach thioredoxins. C. reinhardtii thioredoxin Ch2 can serve as a substrate for E. coli thioredoxin reductase with a lower efficiency when compared to the homologous system. In the presence of dithiothreitol (DTT), the protein is able to catalyze the reduction of porcine insulin. Thioredoxin Ch2 is as efficient as its spinach counterpart in the DTT or light activation of corn NADP-malate dehydrogenase, but it only activates spinach fructose-1, 6-bisphosphatase at very high concentrations. The complete primary structure of the C. reinhardtii thioredoxin Ch2 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin, clostripain, and SV8 protease digestions. It consists of a polypeptide of 106 amino acids (MW 11,808) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. The sequence of the algal thioredoxin Ch2 has been compared to that of thioredoxins from other sources and has the greatest similarity (67%) with the thioredoxin from Anabaena 7119.     

Isomers in thioredoxins of spinach chloroplasts

We have developed a method for the concomitant purification of several components of the ferredoxin/thioredoxin system of spinach chloroplasts. By applying this method to spinach-leaf extract or spinach-chloroplast extract we separated and purified three thioredoxins indigenous to chloroplasts. The three thioredoxins, when reduced, will activate certain chloroplast enzymes such as fructose-1,6-bisphosphatase and NADP-dependent malate dehydrogenase. Fructose-1,6-bisphosphatase is activated by thioredoxin f exclusively. Malate dehydrogenase is activated by thioredoxin mb and thioredoxin mc in a similar way, and it is also activated by thioredoxin f but with different kinetics. All three thioredoxins have very similar relative molecular masses of about 12,000 but distinct isoelectric points of 6.1 (thioredoxin f), 5.2 (thioredoxin mb) and 5.0 (thioredoxin mc). The amino acid composition as well as the C-terminal and N-terminal sequences have been determined for each thioredoxin. Thioredoxin f exhibits clear differences in amino acid composition and terminal sequences when compared with the m-type thioredoxins. Thioredoxin mb and thioredoxin mc, however, are very similar, the only difference being an additional lysine residue at the N-terminus of thioredoxin mb. Amino acid analyses, terminal sequences, immunological tests and the activation properties of the thioredoxins support our conclusion that thioredoxins mb and mc are N-terminal redundant isomers coming from one gene whereas thioredoxin f is a different protein coded by a different gene.     

Glutaredoxins and thioredoxins in plants

During the 70s and 80s two plant thioredoxin systems were identified. The chloroplastic system is composed of a ferredoxin-dependent thioredoxin, with two thioredoxin types (m and f) regulating the activity of enzymes implicated in photosynthetic carbon assimilation. In the cytosol of heterotrophic tissues, an NADP dependent thioredoxin reductase and a thioredoxin (h) were identified. The first plant glutaredoxin was only identified later, in 1994. Our view of plant thioredoxins and glutaredoxins was profoundly modified by the sequencing programs which revealed an unexpected number of genes encoding not only the previously identified disulfide reductases, but also numerous new types. At the same time it became clear that plant genomes encode chloroplastic, cytosolic and mitochondrial peroxiredoxins, suggesting a major role for redoxins in anti-oxidant defense. Efficient proteomics approaches were developed allowing the characterization of numerous thioredoxin target proteins. They are implicated in different aspects of plant life including development and adaptation to environmental changes and stresses. The most important challenge for the next years will probably be to identify in planta which redoxin reduces which target, a question which remains unsolved due to the low specificities of redoxins in vitro and the numerous redundancies which in most cases mask the phenotype of redoxin mutants.     

Crystal structures of two functionally different thioredoxins in spinach chloroplasts

Thioredoxins are small ubiquitous proteins which act as general protein disulfide reductases in living cells. Chloroplasts contain two distinct thioredoxins ( f and m) with different phylogenetic origin. Both act as enzyme regulatory proteins but have different specificities towards target enzymes. Thioredoxin f (Trx f), which shares only low sequence identity with thioredoxin m (Trx m) and with all other known thioredoxins, activates enzymes of the Calvin cycle and other photosynthetic processes. Trx m shows high sequence similarity with bacterial thioredoxins and activates other chloroplast enzymes. The here described structural studies of the two chloroplast thioredoxins were carried out in order to gain insight into the structure/function relationships of these proteins. Crystal structures were determined for oxidized, recombinant thioredoxin f (Trx f-L) and at the N terminus truncated form of it (Trx f-S), as well as for oxidized and reduced thioredoxin m (at 2.1 and 2.3 A resolution, respectively). Whereas thioredoxin f crystallized as a monomer, both truncated thioredoxin f and thioredoxin m crystallized as non-covalent dimers. The structures of thioredoxins f and m exhibit the typical thioredoxin fold consisting of a central twisted five-stranded beta-sheet surrounded by four alpha-helices. Thioredoxin f contains an additional alpha-helix at the N terminus and an exposed third cysteine close to the active site. The overall three-dimensional structures of the two chloroplast thioredoxins are quite similar. However, the two proteins have a significantly different surface topology and charge distribution around the active site. An interesting feature which might significantly contribute to the specificity of thioredoxin f is an inherent flexibility of its active site, which has expressed itself crystallographically in two different crystal forms.