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.     

The ferredoxin-thioredoxin system of a green alga,Chlamydomonas reinhardtii

The components of the ferredoxin-thioredoxin (FT) system of Chlamydomonas reinhardtii have been purified and characterized. The system resembled that of higher plants in consisting of a ferredoxin-thioredoxin reductase (FTR) and two types of thioredoxin, a single f and two m species, m1 and m2. The Chlamydomonas m and f thioredoxins were antigenically similar to their higher-plant counterparts, but not to one another. The m thioredoxins were recognized by antibodies to both higher-plant m and bacterial thioredoxins, whereas the thioredoxin f was not. Chlamydomonas thioredoxin f reacted, although weakly, with the antibody to spinach thioredoxin f. The algal thioredoxin f differed from thioredoxins studied previously in behaving as a basic protein on ion-exchange columns. Purification revealed that the algal thioredoxins had molecular masses (M_rs) typical of thioredoxins from other sources, m1 and m2 being 10700 and f 11 500. Chlamydomonas FTR had two dissimilar subunits, a feature common to all FTRs studied thus far. One, the 13-kDa (similar) subunit, resembled its counterpart from other sources in both size and antigenicity. The other, 10-kDa (variable) sub-unit was not recognized by antibodies to any FTR tested. When combined with spinach, (Spinacia oleracea L.) thylakoid membranes, the components of the FT system functioned in the light activation of the standard target enzymes from chloroplasts, corn (Zea mays L.) NADP-malate dehydrogenase (EC 1.1.1.82) and spinach fructose 1,6-bisphosphatase (EC 3.1.3.11) as well as the chloroplast-type fructose 1,6-bisphosphatase from Chlamydomonas. Activity was greatest if ferredoxin and other components of the FT system were from Chlamydomonas. The capacity of the Chlamydomonas FT system to activate autologous FBPase indicates that light regulates the photosynthetic carbon metabolism of green algae as in other oxygenic photosynthetic organisms.Abbreviations DEAE diethylaminoethyl - ELISA enzyme-linked immunosorption assay - FBPase fructose 1,6-bisphosphatase - Fd ferredoxin - FPLC fast protein liquid chromatography - FTR ferredoxin-thioredoxin reductase - FT system ferredoxin-thioredoxin system - kDa kilodaltons - M_r relative molecular mass - NADP-MDH NADP-malate dehydrogenase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported in part by a grant from the National Aeronautics and Space Administration. We would like to thank Don Carlson and Jacqueline Girard for their assistance with cell cultures.     

Contrasting modes of photosynthetic enzyme regulation in oxygenic and anoxygenic prokaryotes

Enzymes that are regulated by the ferredoxin/thioredoxin system in chloroplasts — fructose-1,6-bisphosphatase (FBPase), sedoheptulose-1,7-bisphosphatase purified from two different types of photosynthetic prokaryotes (cyanobacteria, purple sulfur bacteria) and tested for a response to thioredoxins. Each of the enzymes from the cyanobacterium Nostoc muscorum, an oxygenic organism known to contain the ferredoxin/thioredoxin system, was activated by thioredoxins that had been reduced either chemically by dithiothreitol or photochemically by reduced ferredoxin and ferredoxin-thioredoxin reductase. Like their chloroplast counterparts, N. muscorum FBPase and SBPase were activated preferentially by reduced thioredoxin f. SBPase was also partially activated by thioredoxin m. PRK, which was present in two regulatory forms in N. muscorum, was activated similarly by thioredoxins f and m. Despite sharing the capacity for regulation by thioredoxins, the cyanobacterial FBPase and SBPase target enzymes differed antigenically from their chloroplast counterparts. The corresponding enzymes from Chromatium vinosum, an anoxygenic photosynthetic purple bacterium found recently to contain the NADP/thioredoxin sytem, differed from both those of cyanobacteria and chloroplasts in showing no response to reduced thioredoxin. Instead, C. vinosum FBPase, SBPase, and PRK activities were regulated by a metabolite effector, 5-AMP. The evidence is in accord with the conclusion that thioredoxins function in regulating the reductive pentose phosphate cycle in oxygenic prokaryotes (cyanobacteria) that contain the ferredoxin/thioredoxin system, but not in anoxygenic prokaryotes (photosynthetic purple bacteria) that contain the NADP/thioredoxin system. In organisms of the latter type, enzyme effectors seem to play a dominant role in regulating photosynthetic carbon dioxide assimilation.     

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.     

New evidence for a role for thioredoxin h in germination and seedling development

Thioredoxin of the h-type — earlier linked to the reduction of wheat (Triticum durum Desf. cv. Monroe) endosperm proteins — was converted from an oxidized to a partially reduced state during germination and seedling development. While the abundance of thioredoxin progressively decreased during this period, the availability of reducing equivalents, defined as the product of the relative abundance of thioredoxin and the percent reduction, increased. The amount of the enzyme catalyzing the reduction of thioredoxin h (NADP-thioredoxin reductase) remained constant. The activities of enzymes generating the NADPH needed for the reduction of thioredoxin (glucose 6-phosphate and 6-phosphogluconate dehydrogenases) increased. The level of thioredoxin h in the endosperm appeared to be controlled by the embryo via hormones. Gibberellic acid enhanced the disappearance of thioredoxin, whereas abscisic acid showed the opposite effect. Moreover, uniconazole, an inhibitor of gibberellic acid synthesis, slowed seedling growth and inhibited the disappearance of thioredoxin in a manner reversible by gibberellic acid. The results are consistent with a role for thioredoxin h in initiating the mobilization of nitrogen and carbon needed for germination and seedling development.Abbreviations ABA cis-abscisic acid - DTT dithiothreitol - GA gibberellin - GA₃ gibberellic acid - mBBr monobromobimane - NTR NADP-thioredoxin reductase This work was supported by National Science Foundation grant MCB-9316496. We thank Dr. Yuji Kamiya for advice and the Sumitomo Chemical Co. for a generous sample of uniconazole.     

Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system—NADP—thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.Abbreviations DTNB dithiolbis(2-nitrobenzoic acid) - FBPase fructose-1,6-bisphosphatase - FTR terredoxin-thioredoxin, reductase - NADP-MDH NADP-malate dehydrogenase - NTR NADP-thioredoxin reductase - SDS sodium-dodecyl sulfate     

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     

The complex regulation of ferredoxin/thioredoxin-related genes by light and the circadian clock

The biochemical properties of the ferredoxin/thioredoxin transduction pathway regulating the activity of key carbon-fixation enzymes through post-translational modifications are well characterized but little is known about the regulation of the different genes. In the present study, we investigated in Chlamydomonas reinhardtii the regulation of the expression of ferredoxin, thioredoxin m, ferredoxin-NADP reductase, phosphoribulokinase, as well as that of cytosolic thioredoxin h, the function of which is still largely unknown. The effects of light, the circadian clock and active cell division were investigated by northern blotting. The five genes were found to be regulated by light and the circadian clock but with different kinetics and amplitudes. This leads for the first time to the proposal that an extra-chloroplastic thioredoxin is possibly implicated in light and/or circadian-related processes. An interplay between several light-transduction pathways in controlling the expression of the genes is suggested by the expression studies and the theoretical analysis of the promoters. Received: 2 December 1998 / Accepted: 19 March 1999     

Isolation and characterization of thioredoxin h from poplar xylem

Redox-dependent regulation based on disulphide/dithiol exchange reactions has been extensively studied in herbaceous plants, but up to now, there is no information concerning these systems in trees. Based on existing ESTs, a cDNA coding for a thioredoxin h has been isolated from a xylem poplar cDNA library. The nucleotidic sequence of poplar thioredoxin h displays significant homology to other thioredoxins h isolated from plants. It shows a variation in the active site with the sequence WCPPC instead of the more canonical WCGPC sequence found in most thioredoxins. The cDNA sequence 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 and purified from Escherichia coli cells with a very high yield. Several of the physical and kinetic characteristics of this redox protein are described and found to be similar to other thioredoxin h. On the other hand, its stability to heat denaturation, is very different from those of other thioredoxins h characterized so far.     

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.     

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.     

Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale

The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale. Received: 19 September 1996 / Accepted: 21 May 1997     

The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis.

Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene.     

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.     

Isolation of two thioredoxins from the cyanobacterium Synechococcus 6301

Two different thioredoxins designated as thioredoxin A and B have been isolated from the cyanobacterium Synechococcus 6301. Methods for large scale purification of these thioredoxins were developed. Thioredoxin B has been purified to homogeneity; it has a molecular weight of 11,800 and an isoelectric point of 4.6. The following K _m data were obtained for this thioredoxin; a) in the PAPS-sulfotransferase assay of Synechococcus 6301: 10.7 M; b) in the fructose-1-6-bisphosphatase assay of Synechococcus 6301: 1.7 M; c) in the APS-sulfotransferase assay of Chroococcidiopsis 7203: 5.4M. Thioredoxin A has an isoelectric point of 4.1 and it is active in the PAPS-sulfotransferase and fructose-1-6-bisphosphatase of Synechococcus 6301; it is not active in the APS-sulfotransferase of Chroococcidiopsis 7203.Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

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.