Identification and characterization of a third thioredoxin h in poplar

Three full-length sequences encoding thioredoxin h have been isolated in a leaf/root of Populus trichocarpa cv. Trichobel expressed sequence tag (EST) library. One of these, popCXXS1 exhibits the nontypical active site CXXS homologous to atCXXS1. The second one, named popTrxh4, is related to atTrxh9 which forms with several other plant thioredoxin h a distinct subgroup of thioredoxins h. The third one, named popTrxh3, displays 66% identity and also a high degree of homology (81%) vs. the previously described popTrxh1. Nevertheless, the active sites of both proteins differ, since the active site of popTrxh1 (WCPPC) is a variant of the canonical WCGPC found in popTrxh3. The cDNA sequence of popTrxh3 has been introduced in an expression plasmid (pET3d) in order to express the corresponding recombinant polypeptide. The protein has been expressed to a high level, purified from Escherichia coli cells with a high yield and its catalytic properties compared to popTrxh1. Furthermore, two mutants, popTrxh1P40G and popTrxh3G41P, have been engineered in order to explore the importance of the active site residues in the thioredoxin h catalytic properties. The results are discussed in relation with known biochemical properties of thioredoxins h.     

The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed

A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h₂) was isolated using thioredoxin h₁ cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 by respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h₁ (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h₁ and h₂, do not have any signal peptides and are most probably cytoplasmic. Using the 3′ regions of the mRNAs, two probes specific for thioredoxins h₁ and h₂ have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h₁ and a 4.5 kb fragment encodes h₂. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h₂ is present in N. sylvestris and N. tomentosiformis but that thioredoxin h₁ is absent from both putative ancestors. Thus, the thioredoxin h₁ gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells. Thioredoxin h₁ is always expressed at a lower level than h₂ in tobacco plants. In contrast, the thioredoxin hl gene is abundantly expressed in freshly isolated protoplasts, while h₂ mRNAs are not detectable.     

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     

Isolation of a chicken thioredoxin cDNA clone. Thioredoxin mRNA is differentially expressed in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts

We have isolated a cDNA clone for chicken thioredoxin by differential screening of a cDNA library. The protein product which this clone encodes is very similar to other thioredoxins, and it displays thioredoxin activity when expressed in Escherichia coli. This clone represents the first metazoan thioredoxin for which the protein or nucleic acid sequence is known. Comparison of the chicken thioredoxin protein sequence with those from bacteria and plants indicates structural features that appear to be essential for activity. Transformation of chicken embryo fibroblasts by Rous sarcoma virus elevates the level of thioredoxin mRNA whereas the level of thioredoxin mRNA in a nonproliferative tissue (brain) is much lower than in chicken embryo fibroblasts.     

The Nicotiana tabacum genome encodes two cytoplasmic thioredoxin genes which are differently expressed

A Nicotiana tabacum thioredoxin h gene (EMBL Accession No. Z11803) encoding a new thioredoxin (called h₂) was isolated using thioredoxin h₁ cDNA (X58527), and represents the first thioredoxin h gene isolated from a higher plant. It encodes a polypeptide of 118 amino acids with the conserved thioredoxin active site Trp-Cys-Gly-Pro-Cys. This gene comprises two introns which have lengths of 1071 and 147 by respectively, and three exons which encode peptides of 29, 41 and 48 amino acids, respectively. This thioredoxin h shows 66% identity with the amino acid sequence of thioredoxin h₁ (X58527) and only around 35% with the choroplastic thioredoxins. The two thioredoxins, h₁ and h₂, do not have any signal peptides and are most probably cytoplasmic. Using the 3 regions of the mRNAs, two probes specific for thioredoxins h₁ and h₂ have been prepared. Southern blot analysis shows that thioredoxin sequences are present in only two genomic EcoRI fragments: a 3.3 kb fragment encodes h₁ and a 4.5 kb fragment encodes h₂. Analysis of the ancestors of the allotetraploid N. tabacum shows that thioredoxin h₂ is present in N. sylvestris and N. tomentosiformis but that thioredoxin h₁ is absent from both putative ancestors. Thus, the thioredoxin h₁ gene has probably been recently introduced in to N. tabacum as a gene of agronomic importance, or linked to such genes. Northern blot analysis shows that both genes are expressed in N. tabacum, mostly in organs or tissues that contain growing cells. Thioredoxin h₁ is always expressed at a lower level than h₂ in tobacco plants. In contrast, the thioredoxin hl gene is abundantly expressed in freshly isolated protoplasts, while h₂ mRNAs are not detectable.     

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.     

An f-type thioredoxin fromArabidopsis thaliana Leaves

Thioredoxin, a small redox protein with an active site disulfide/dithiol, is ubiquitous in bacteria, plants, and animals and functions as a reducing agent and modulator of enzyme activity. A thioredoxin has been purified to electrophoretic homogeneity from the leaves ofArabidopsis thaliana using procedures such as DE-52 ion exchange chromatography, Sephadex G-50 gel filtration, Q-Sepharose ion exchange chromatography, and DEAE-Sephadex A-25 chromatography. The purified thioredoxin was determined to be a single band on SDS-PAGE, and its molecular weight was estimated to be 21 KDa, which was much larger than those of most other known thioredoxins. It was proved to be an f-type thioredoxin, since it could activate fructose-l,6-bisphosphatase, but it could not activate NADP⁺-malate dehydrogenase. As a protein disulfide reductase, it could reduce the disulfide bonds contained in insulin. As a substrate, it showed a Km value of 20.2 μM onEscherichia coli thioredoxin reductase, and it had an optimal pH of 8.0. The molecular weight of the purified f-type thioredoxin is not consistent with those of the five divergent h-type thioredoxins already identified by cDNA cloning. The purified f-type thioredoxin is the first example isolated fromA. thaliana.     

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.     

Activities of two dissimilar thioredoxins from the cyanobacterium Anabaena sp. strain PCC 7120.

Thioredoxin is a small redox protein that functions as a reducing agent and modulator of enzyme activity. A gene for an unusual thioredoxin was previously isolated from the cyanobacterium Anabaena sp. strain PCC 7120 and cloned and expressed in Escherichia coli. However, the protein could not be detected in Anabaena cells (J. Alam, S. Curtis, F. K. Gleason, M. Gerami-Nejad, and J. A. Fuchs, J. Bacteriol. 171:162-171, 1989). Polyclonal antibodies to the atypical thioredoxin were prepared, and the protein was detected by Western immunoblotting. It occurs at very low levels in extracts of Anabaena sp. and other cyanobacteria. No antibody cross-reaction was observed in extracts of eukaryotic algae, plants, or eubacteria. The anti-Anabaena thioredoxin antibodies did react with another unusual thioredoxin-glutaredoxin produced by bacteriophage T4. Like the T4 protein and other glutaredoxins, the unusual cyanobacterial thioredoxin can be reduced by glutathione. The Anabaena protein can also activate enzymes of carbon metabolism and has some functional similarity to spinach chloroplast thioredoxin f. However, it shows only 23% amino acid sequence identity to the spinach chloroplast protein and appears to be distantly related to other thioredoxins. The data indicate that cyanobacteria, like plant chloroplasts, have two dissimilar thioredoxins. One is related to the more common protein found in other prokaryotes, and the other is an unusual thioredoxin that can be reduced by glutathione and may function in glucose catabolism.     

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.     

Isolation and characterization of thioredoxin f from the filamentous cyanobacterium, Anabaena sp. 7119

Two thioredoxin fractions had previously been reported to occur in Anabaena 7119 by Buchanan and co-workers (Yee, B. C., dela Torre, A., Crawford, N. A., Lara, C., Carlson, D. E., and Buchanan, B. B. (1981) Arch. Microbiol. 130, 14-18). These proteins were detected by their ability to activate spinach fructose-1,6-bisphosphatase (Fru-P2-ase). The partially purified proteins resembled similar thioredoxins found in spinach chloroplasts and were designated thioredoxin f (Tf) for the fraction most effective in activating spinach Fru-P2-ase and thioredoxin m (Tm) for the fraction most effective in activating spinach NADPH-malate dehydrogenase. Using the assay system of Yee and co-workers, we were able to separate and purify to homogeneity two thioredoxin fractions from Anabaena extracts. Tm corresponded to the thioredoxin fraction we had isolated and studied previously (Gleason, F. K., and Holmgren, A. (1981) J. Biol. Chem. 256, 8301-8309). The other fraction, Tf, was characterized further. Unlike the thioredoxins found in higher plants, the cyanobacterial thioredoxins do not appear to be related. Anabaena thioredoxin f has a Mr = 25,500 as compared to the more usual Mr = 12,000 for Tm. From a comparison of the amino acid composition, Tf is not obviously a dimer or otherwise related to Tm. Tf has one active center cystine disulfide. Anabaena Tf activates spinach Fru-P2-ase very efficiently but has very little activity with spinach malate dehydrogenase. Anabaena Tf, unlike Tm, does not reduce the homologous ribonucleotide reductase. Anabaena Tf also does not activate a partially purified preparation of Anabaena Fru-P2-ase. We conclude that the cyanobacterial Tf is a unique protein with no structural or functional properties in common with other thioredoxins.     

A novel plasma membrane-bound thioredoxin from soybean

Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.     

Complementary DNA cloning of poplar bark storage protein and control of its expression by photoperiod

Bark storage proteins accumulate in the bark of many woody plants during autumn and winter. In poplar (Populus deltoides Bartr. ex Marsh), the accumulation of the 32-kilodalton bark storage protein is controlled by photoperiod. We have isolated a full-length cDNA encoding for the poplar 32-kilodalton bark storage protein and determined its nucleotide sequence. The derived amino acid sequence shows that poplar bark storage protein is rich in serine, leucine, phenylalanine, and lysine. Poplar bark storage protein is similar to the poplar wound-induced cDNA clone 4 and clone 16 (TJ Parsons, HD Bradshaw, MP Gordon [1989] Proc Natl Acad Sci USA 86: 7895-7899). DNA gel blot analysis suggests that poplar bark storage protein is encoded by a multigene family of about five genes. Poplar plants grown in long days contained low levels of mRNA for the bark storage protein. Exposure to short days resulted in an increase in bark storage protein mRNA within 7 days. After 21 days of short day exposure, high levels of mRNA were detected. The accumulation of bark storage protein mRNA in response to short days was also observed in plants exposed to natural shortening daylengths. Our results indicate that the accumulation of poplar bark storage protein mRNA is controlled by photoperiod. This finding will provide a useful system for investigating photoperiodism in woody plants.     

Association of a thioredoxin-like protein with chloroplaast coupling factor (CF1).

The δ subunit isolated from chloroplast coupling factor (CF₁) preparations partially replaced thioredoxin in the dithiothreitol-linked activation of chloroplast fructose 1,6-bisphosphatase. The δ subunit fraction also stimulated the dithiothreitol-dependent ATPase of heated CF₁ in a manner analogous to that obseryed with each of the three thioredoxins isolated from spinach leaves (thioredoxins f, m, and c). The δ subunit used in most of these experiments was obtained from CF₁ that had been isolated by a newly devised procedure based on acid precipitation.     

Thioredoxins in<Emphasis Type="Italic">Arabidopsis</Emphasis> and other plants

Regulation of disulfide dithiol exchange has become increasingly important in our knowledge of plant life. Initially discovered as regulators of light-dependent malate biosynthesis in the chloroplast, plant thioredoxins are now implicated in a large panel of reactions related to metabolism, defense and development. In this review we describe the numerous thioredoxin types encoded by the Arabidopsis genome, and provide evidence that they are present in all higher plants. Some results suggest cross-talk between thioredoxins and glutaredoxins, the second family of disulfide dithiol reductase. The development of proteomics in plants revealed an unexpectedly large number of putative target proteins for thioredoxins and glutaredoxins. Nevertheless, we are far from a clear understanding of the actual function of each thioredoxin in planta. Although hampered by functional redundancies between genes, genetic approaches are probably unavoidable to define which thioredoxin interacts with which target protein and evaluate the physiological consequences.     

Solution structure of thioredoxin h1 from Arabidopsis thaliana

Present in virtually every species, thioredoxins catalyze disulfide/dithiol exchange with various substrate proteins. While the human genome contains a single thioredoxin gene, plant thioredoxins are a complex protein family. A total of 19 different thioredoxin genes in six subfamilies has emerged from analysis of the Arabidopsis thaliana genome. Some function specifically in mitochondrial and chloroplast redox signaling processes, but target substrates for a group of eight thioredoxin proteins comprising the h subfamily are largely uncharacterized. In the course of a structural genomics effort directed at the recently completed A. thaliana genome, we determined the structure of thioredoxin h1 (At3g51030.1) in the oxidized state. The structure, defined by 1637 NMR-derived distance and torsion angle constraints, displays the conserved thioredoxin fold, consisting of a five-stranded beta-sheet flanked by four helices. Redox-dependent chemical shift perturbations mapped primarily to the conserved WCGPC active-site sequence and other nearby residues, but distant regions of the C-terminal helix were also affected by reduction of the active-site disulfide. Comparisons of the oxidized A. thaliana thioredoxin h1 structure with an h-type thioredoxin from poplar in the reduced state revealed structural differences in the C-terminal helix but no major changes in the active site conformation.