Thioredoxin Gene Research Articles

Molecular Identification of Two Thioredoxin Genes From Grapholita molesta and Their Function in Resistance to Emamectin Benzoate.

Thioredoxins (Trxs), a member of the thioredoxin system, play crucial roles in maintaining intracellular redox homeostasis and protecting organisms against oxidative stress. In this study, we cloned and characterized two genes, GmTrx2 and GmTrx-like1, from Grapholita molesta. Sequence analysis showed that GmTrx2 and GmTrx-like1 had highly conserved active sites CGPC and CXXC motif, respectively, and shared high sequence identity with selected insect species. The quantitative real-time polymerase chain reaction results revealed that GmTrx2 was mainly detected at first instar, whereas GmTrx-like1 was highly concentrated at prepupa day. The transcripts of GmTrx2 and GmTrx-like1 were both highly expressed in the head and salivary glands. The expression levels of GmTrx2 and GmTrx-like1 were induced by low or high temperature, E. coli, M. anisopliae, H2O2, and pesticides (emamectin benzoate). We further detected interference efficiency of GmTrx2 and GmTrx-like1 in G. molesta larvae and found that peroxidase capacity, hydrogen peroxide content, and ascorbate content all increased after knockdown of GmTrx2 or GmTrx-like1. Furthermore, the hydrogen peroxide concentration was increased by emamectin benzoate and the sensitivity for larvae to emamectin benzoate was improved after GmTrx2 or GmTrx-like1 was silenced. Our results indicated that GmTrx2 and GmTrx-like1 played vital roles in protecting G. molesta against oxidative damage and also provided the theoretical basis for understanding the antioxidant defense mechanisms of the Trx system in insects.

Effects and related mechanism of overexpression of human thioredoxin on the inflammatory response in mice with viral myocarditis

Objective: To observe the effects of recombinant adenovirus with human thioredoxin (hTRX) on the inflammatory response in mice with viral myocarditis and explore the related mechanism. Methods: Sixty Balb/c male mice were randomly divided into control group, myocarditis group, and hTRX group according to the random number table (n=20 each group). The myocarditis group and hTRX group were injected with 100 TCID(50) Coxackie virus B3 (0.1 ml) in the abdomen and control group were injected with saline. Two days before the viral injection, the hTRX group were injected with recombinant adenovirus vector coding the human thioredoxin gene by pericardial puncture and the control group and myocarditis group were injected with recombinant adenovirus vector without coding gene by pericardial puncture, all these mice were killed and hearts were removed 7 days later. The morphology of myocardial tissue in each group was detected by HE staining and the ultrastructure changes by electron microscope. The protein expressions of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and NF-κB were detected by ELISA and Western blot. Immunohistochemical staining was performed to observe the protein expression levels of thioredoxin. Results: Necrosis of myocardial cells and a small amount of cell infiltration were found in the myocarditis group and necrosis and cell infiltration were significantly reduced in the hTRX group and no myocardial lesion was found in control group on HE stained sections. Electron microscope examination evidenced cell swelling and dissolved myofilament, vacuoles degeneration in mitochondria in the myocarditis group. These changes were significantly reduced in the hTRX group. There was no myocardial lesion in control group. The protein expression of TNF-α, IL-1β and NF-κB were significantly upregulated in myocarditis group than in control group (all P<0.01). The protein expression of TNF-α, IL-1β and NF-κB were significantly downregulated in hTRX group than in myocarditis group (all P<0.01). Immunohistochemical staining showed that protein expression of hTRX was higher in hTRX group than in myocarditis group (P<0.01). Conclusion: Recombinant adenovirus hTRX can attenuate cardiac injury in mice with acute myocarditis via inhibiting the inflammatory response and downregulating the expression of TNF-α, IL-1β and NF-κB.

Cloning and functional characterization of thioredoxin genes from large yellow croaker Larimichthys crocea

Thioredoxin(Trx)with a redox-active disulfide/dithiol in the active site, is an ubiquitous disulfide reductase majorly responsible for maintaining the balance of reactive oxygen species. In this study, the complete thioredoxin-like protein 1 (designated as LcTrx) was cloned from large yellow croaker Larimichthys crocea through rapid amplification of cDNA ends. The full-length cDNA of LcTrx was 1295 bp in length containing a 131 bp 5′ untranslated region (UTR) ,a 3′UTR of 294bp with a poly (A) tail, and an 870 bp open reading frame (ORF) encoding a polypeptide of 289 amino acids. Protein sequence analysis revealed that LcTrx contains the evolutionarily conserved redox motif CRPC (Cys-Arg-Pro-Cys-). Multiple alignments revealed that LcTrx is highly identical to Trx from other organisms, especially in the CRPC motifs. The recombinant LcTrx showed obvious insulin reduction activity in vitro. The LcTrx transcripts were constitutively expressed in all examined tissues with the highest levels found in the muscles and the lowest in the head kidney. Results of Vibrio parahaemolyticus infection experiment showed that the expression levels of LcTrx were tissue and time dependent. In the liver and kidney, LcTrx was down-regulated both at 12 h and 48 h post-infection. In contrast, LcTrx showed induced expression in the spleen and head kidney at same post-infection time points. The different responses to pathogen stimulation indicated the diversified physiological function of LcTrx in the four examined tissues.

Inhibition of thioredoxin activates mitophagy and overcomes adaptive bortezomib resistance in multiple myeloma

BackgroundAlthough current chemotherapy using bortezomib (Velcade) against multiple myeloma in adults has achieved significant responses and even remission, a majority of patients will develop acquired resistance to bortezomib. Increased thioredoxin level has been reported to be associated with carcinogenesis; however, the role of thioredoxin in bortezomib drug resistance of myeloma remains unclear.MethodsWe generated several bortezomib-resistant myeloma cell lines by serially passaging with increased concentrations of bortezomib over a period of 1.5 years. Thioredoxin expression was measured by real-time PCR and western blot.ResultsThe role of thioredoxin in the survival of bortezomib-resistant myeloma cells was determined by specific shRNA knockdown in vitro and in vivo. Thioredoxin inhibitor (PX12) was used to determine the effectiveness of thioredoxin inhibition in the treatment of bortezomib-resistant myeloma cells. The effect of thioredoxin inhibition on mitophagy induction was examined. The correlation of thioredoxin expression with patient overall survival was interrogated. Thioredoxin expression was significantly upregulated in bortezomib-resistant myeloma cells and the change correlated with the increase of bortezomib concentration. Thioredoxin gene knockdown using specific shRNA sensitized bortezomib-resistant myeloma cells to bortezomib efficiency in vitro and in vivo. Similarly, pharmacological inhibition with PX12 inhibited the growth of bortezomib-resistant myeloma cells and overcame bortezomib resistance in vitro and in vivo. Furthermore, inhibition of thioredoxin resulted in the activation of mitophagy and blockage of mitophagy prevented the effects of PX12 on bortezomib-resistant myeloma cells, indicating that mitophagy is the important molecular mechanism for the induction of cell death in bortezomib-resistant myeloma cells by PX12. Moreover, inhibition of thioredoxin resulted in downregulation of phosphorylated mTOR and ERK1/2. Finally, thioredoxin was overexpressed in primary myeloma cells isolated from bortezomib-resistant myeloma patients and overexpression of thioredoxin correlated with poor overall survival in patients with multiple myeloma.ConclusionsOur findings demonstrated that increased thioredoxin plays a critical role in bortezomib resistance in multiple myeloma through mitophagy inactivation and increased mTOR and ERK1/2 phosphorylation. Thioredoxin provides a potential target for clinical therapeutics against multiple myeloma, particularly for bortezomib-resistant/refractory myeloma patients.

Expression of genes and proteins of multidrug resistance in gastric cancer cells treated with resveratrol.

Multidrug resistance (MDR) is a notable problem in the use of chemotherapy. Therefore, studies aimed at identifying substances capable of overcoming resistance of cancer cells are required. Examples of these compounds are polyphenols, including resveratrol, that exert a range of various biological activities. The aim of the present study was to demonstrate the effect of 3,5,4'-trihydroxy-trans-stilbene (resveratrol) on the expression of ATP binding cassette subfamily B member 1, Annexin A1 (ANXA1) and thioredoxin (TXN) genes, and the proteins encoded by these genes, which are associated with MDR. The experiments were performed in human gastric cancer cell lines EPG85-257RDB (RDB) and EPG85-257RNOV (RNOV), which are resistant to daunorubicin and mitoxantrone, respectively, in addition to EPG85-257P (control), which is sensitive to cytostatic drugs. Cells were treated with 30 or 50 µM resveratrol for 72 h and changes in the expression levels of the genes were analysed with the use of a reverse transcription-quantitative polymerase chain reaction. The cellular levels of P-glycoprotein (P-gp), ANXA1 and TXN were evaluated using immunofluorescence and western blot analysis. Resveratrol in both concentrations has been shown to have a statistically significant influence on expression of the mentioned genes, compared with untreated cells. In RDB cells, resveratrol reduced the expression level of all analyzed genes, compared with untreated cells. Similar results at the protein level were obtained for P-gp and TXN. In turn, in the RNOV cell line, resveratrol reduced TXN expression at mRNA and protein levels, compared with untreated cells. The results of the present study indicate that resveratrol may reduce the resistance of cancer cells by affecting the expression of a number of the genes and proteins associated with MDR.

Comparison of MAPK and thioredoxin gene expression in wheat seedlings exposed to silver nitrate and silver nanoparticle

The extensive use of heavy metals and nanoparticles (NPs) has led to their release into the environment that might have negative impacts on both organisms and the environment. In this study, the molecular responses of wheat seedlings to silver nitrate and silver nanoparticles (AgNPs) were assessed by transcript accumulation analysis of genes coding for products potentially involved in heavy metal tolerance. A quantitative Real-time PCR experiment was performed with MAPK (Mitogen-activated protein kinase) and thioredoxin genes using RNA isolated from wheat shoots treated for 0, 2, 6, 12 and 24 h with AgNO3 and AgNPs at 100 mg-1L concentration. Results indicated that stressful conditions led to the antioxidant responses of wheat seedlings that could be reflected as changes in MAPK and thioredoxin gene transcripts. The genes expression patterns were slightly different. The expression of these genes in response to both treatments was high at the beginning of the stress and was decreased with time. Our results showed the effects of AgNO3 treatment were faster than AgNPs. We found that wheat seedlings might develop different strategies to cope with AgNO3 and AgNPs toxicity with change in the expression of MAPK and thioredoxin heavy metal-related genes.

Stress response of a clinical Enterococcus faecalis isolate subjected to a novel antimicrobial surface coating

Emerging antibiotic resistance among pathogenic bacteria, paired with their ability to form biofilms on medical and technical devices, represents a serious problem for effective and long-term decontamination in health-care environments and gives rise to an urgent need for new antimicrobial materials. Here we present the impact of AGXX®, a novel broad-spectrum antimicrobial surface coating consisting of micro-galvanic elements formed by silver and ruthenium, on the transcriptome of Enterococcus faecalis. A clinical E. faecalis isolate was subjected to metal stress by growing it for different periods in presence of the antimicrobial coating or silver-coated steel meshes. Subsequently, total RNA was isolated and next-generation RNA sequencing was performed to analyze variations in gene expression in presence of the antimicrobial materials with focus on known stress genes. Exposure to the antimicrobial coating had a large impact on the transcriptome of E. faecalis. After 24min almost 1/5 of the E. faecalis genome displayed differential expression. At each time-point the cop operon was strongly up-regulated, providing indirect evidence for the presence of free Ag+-ions. Moreover, exposure to the antimicrobial coating induced a broad general stress response in E. faecalis. Genes coding for the chaperones GroEL and GroES and the Clp proteases, ClpE and ClpB, were among the top up-regulated heat shock genes. Differential expression of thioredoxin, superoxide dismutase and glutathione synthetase genes indicates a high level of oxidative stress. We postulate a mechanism of action where the combination of Ag+-ions and reactive oxygen species generated by AGXX® results in a synergistic antimicrobial effect, superior to that of conventional silver coatings.

Synergistic effect of thioredoxin and its reductase from Kluyveromyces marxianus on enhanced tolerance to multiple lignocellulose-derived inhibitors

BackgroundMultiple lignocellulose-derived inhibitors represent great challenges for bioethanol production from lignocellulosic materials. These inhibitors that are related to the levels of intracellular reactive oxidative species (ROS) make oxidoreductases a potential target for an enhanced tolerance in yeasts.ResultsIn this study, the thioredoxin and its reductase from Kluyveromyces marxianus Y179 was identified, which was subsequently achieved over-expression in Saccharomyces cerevisiae 280. In spite of the negative effects by expression of thioredoxin gene (KmTRX), the thioredoxin reductase (KmTrxR) helped to enhance tolerance to multiple lignocellulose-derived inhibitors, such as formic acid and acetic acid. In particular, compared with each gene expression, the double over-expression of KmTRX2 and KmTrxR achieved a better ethanol fermentative profiles under a mixture of formic acid, acetic acid, and furfural (FAF) with a shorter lag period. At last, the mechanism that improves the tolerance depended on a normal level of intracellular ROS for cell survival under stress.ConclusionsThe synergistic effect of KmTrxR and KmTRX2 provided the potential possibility for ethanol production from lignocellulosic materials, and give a general insight into the possible toxicity mechanisms for further theoretical research.

Jab1/Csn5-Thioredoxin Signaling in Relapsed Acute Monocytic Leukemia under Oxidative Stress.

Purpose: High levels of ROS and ineffective antioxidant systems contribute to oxidative stress, which affects the function of hematopoietic cells in acute myeloid leukemia (AML); however, the mechanisms by which ROS lead to malignant transformation in relapsed AML-M5 are not completely understood. We hypothesized that alterations in intracellular ROS would trigger AML-M5 relapse by activating the intrinsic pathway.Experimental Design: We studied ROS levels and conducted c-Jun activation domain-binding protein-1 (JAB1/COPS5) and thioredoxin (TRX) gene expression analyses with blood samples obtained from 60 matched AML-M5 patients at diagnosis and relapse and conducted mechanism studies of Jab1's regulation of Trx in leukemia cell lines.Results: Our data showed that increased production of ROS and a low capacity of antioxidant enzymes were characteristics of AML-M5, both at diagnosis and at relapse. Consistently, increased gene expression levels of TRX and JAB1/COPS5 were associated with low overall survival rates in patients with AML-M5. In addition, stimulating AML-M5 cells with low concentrations of hydrogen peroxide led to increased Jab1 and Trx expression. Consistently, transfection of ectopic Jab1 into leukemia cells increased Trx expression, whereas silencing of Jab1 in leukemia cells reduced Trx expression. Mechanistically, Jab1 interacted with Trx and stabilized Trx protein. Moreover, Jab1 transcriptionally regulated Trx. Furthermore, depletion of Jab1 inhibited leukemia cell growth both in vitro and in vivoConclusions: We identified a novel Jab1-Trx axis that is a key cellular process in the pathobiologic characteristics of AML-M5. Targeting the ROS/Jab1/Trx pathway could be beneficial in the treatment of AML-M5. Clin Cancer Res; 23(15); 4450-61. ©2017 AACR.

Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum

Corynebacterium glutamicum is an important industrial producer of various amino acids and other metabolites. The C. glutamicum genome encodes seven sigma subunits (factors) of RNA polymerase: the primary sigma factor SigA (σA), the primary-like σB and five alternative sigma factors (σC, σD, σE, σH and σM). We have developed in vitro and in vivo methods to assign particular sigma factors to individual promoters of different classes. In vitro transcription assays and measurements of promoter activity using the overexpression of a single sigma factor gene and the transcriptional fusion of the promoter to the gfpuv reporter gene enabled us to reliably define the sigma factor dependency of promoters. To document the strengths of these methods, we tested examples of respective promoters for each C. glutamicum sigma factor. Promoters of the rshA (anti-sigma for σH) and trxB1 (thioredoxin) genes were found to be σH-dependent, whereas the promoter of the sigB gene (sigma factor σB) was σE- and σH-dependent. It was confirmed that the promoter of the cg2556 gene (iron-regulated membrane protein) is σC-dependent as suggested recently by other authors. The promoter of cmt1 (trehalose corynemycolyl transferase) was found to be clearly σD-dependent. No σM-dependent promoter was identified. The typical housekeeping promoter P2sigA (sigma factor σA) was proven to be σA-dependent but also recognized by σB. Similarly, the promoter of fba (fructose-1,6-bisphosphate aldolase) was confirmed to be σB-dependent but also functional with σA. The study provided demonstrations of the broad applicability of the developed methods and produced original data on the analyzed promoters.

Expression of some Genes in Response to Cadmium Stress in &lt;i&gt;Triticum aestivum&lt;/i&gt;

Heavy metal toxicity has become a universal threat to all life forms, including plants. The main purpose of this study was to identify the gene expression profiling of MAPK, Thioredoxin, and MnSOD genes in wheat seedlings as affected by cadmium treatment. For this experiment, the quantitative Real-Time PCR on RNA isolated from shoots of wheat exposed to CdCl2 at a concentration of 100 mg/L was used. Results showed that in wheat seedling that exposed to cadmium stress for six days of beginning constant cadmium stress, Thioredoxin gene expression showed a large rise compared with the control sample, MnSOD gene expression increased compared with non-treated wheat seedling at the same times, but unlike the Thioredoxin and MnSOD genes, MAPK gene expression has no significant changes. Of course, it is possible that other times of beginning treatments (instead of six days) cause a change in this gene expression.

Vaccination with a live attenuated Acinetobacter baumannii deficient in thioredoxin provides protection against systemic Acinetobacter infection

Multi-drug resistant Acinetobacter baumannii (MDR-Ab), an opportunistic pathogen associated with nosocomial and combat related infections, has a high mortality due to its virulence and limited treatment options. Deletion of the thioredoxin gene (TrxA) from a clinical isolate of MDR-Ab resulted in a 100-fold increase in 50% lethal dose (LD50) in a systemic challenge murine model. Thus, we investigated the potential use of this attenuated strain as a live vaccine against MDR-Ab. Mice were vaccinated by subcutaneous (s.c.) injection of 2×105 CFU of the ΔtrxA mutant, boosted 14days later with an equivalent inoculum, and then challenged 30days post-vaccination by i.p. injection with 10 LD50 of the wild type (WT) Ci79 strain. Efficacy of vaccination was evaluated by monitoring MDR-Ab specific antibody titers and cytokine production, observing pathology and organ burdens after WT challenge, and measuring levels of serum pentraxin-3, a molecular correlate of A. baumannii infection severity, before and after challenge. Mice vaccinated with ΔtrxA were fully protected against the lethal challenge of WT. However, minimal immunoglobulin class switching was observed with IgM predominating. Spleens harvested from vaccinated mice exhibited negligible levels of IL-4, IFN-γ and IL-17 production when stimulated with UV-inactivated WT Ci79. Importantly, tissues obtained from vaccinated mice displayed reduced pathology and organ burden compared to challenged non-vaccinated mice. Additionally, serum pentraxin-3 concentrations were not increased 24h after challenge in vaccinated mice, correlating with reduction of WT MDR-Ab infection in ΔtrxA immunized mice. Furthermore, passive immunization with ΔtrxA-immune sera provided protection against lethal systemic Ci79 challenge. Collectively, the defined live attenuated ΔtrxA strain is a vaccine candidate against emerging MDR Acinetobacter infection.

Expression of some Genes in Response to Cadmium Stress in &lt;i&gt;Triticum aestivum&lt;/i&gt;

Heavy metal toxicity has become a universal threat to all life forms, including plants. The main purpose of this study was to identify the gene expression profiling of MAPK, Thioredoxin, and MnSOD genes in wheat seedlings as affected by cadmium treatment. For this experiment, the quantitative Real-Time PCR on RNA isolated from shoots of wheat exposed to CdCl2 at a concentration of 100 mg/L was used. Results showed that in wheat seedling that exposed to cadmium stress for six days of beginning constant cadmium stress, Thioredoxin gene expression showed a large rise compared with the control sample, MnSOD gene expression increased compared with non-treated wheat seedling at the same times, but unlike the Thioredoxin and MnSOD genes, MAPK gene expression has no significant changes. Of course, it is possible that other times of beginning treatments (instead of six days) cause a change in this gene expression.

A thioredoxin from antarctic microcrustacean (Euphausia superba): Cloning and functional characterization

Thioredoxins, with a dithiol/disulfide active site (CGPC) are major highly conserved and ubiquitous proteins that are involved in protecting organisms against various oxidative stresses. In the present study, a novel thioredoxin gene was identified in antarctic microcrustacean, Euphausia superba (designated as EsTrx1). The full-length cDNA sequences of EsTrx1 was of 621 bp, containing a 5′ untranslated region (UTR) of 45 bp, a 3′ UTR of 276 bp and an open reading frame (ORF) of 303 bp encoding a putative protein of 100 amino acids. The predicted molecular weight of EsTrx1 was 11.08 kDa and the theoretical isoelectric point was 4.51. Multiple sequence alignment indicated that the EsTrx1 possessed conserved CGPC redox-active site. EsTrx1 shared 68.6% similarity with the Chinese mitten crab (Eriocheir sinensis) Trx1. The predicted three-dimensional structure of EsTrx1 consisted of a central core of a four-stranded β-sheet and four flanking α-helices. The high similarity of EsTrx1 with Trx1s from other animals together with the phylogenetic analysis indicated that EsTrx1 could be a novel member of Trx1 sub-family. In order to elucidate its biological functions, the recombinant EsTrx1 was constructed and expressed in Escherichia coli BL21 (DE3). Experiments demonstrated that the rEsTrx1 fusion protein possessed the expected redox activity in enzymatic analysis, and be more potent than GSH in antioxidant capacity. These results together indicated that EsTrx1 could be involved in the oxidative stress response of E. superba.

New Antioxidant Genes from an Anhydrobiotic Insect: Unique Structural Features in Functional Motifs of Thioredoxins

Polipedilum vanderplanki is the most complex known organism able to survive body desiccation via entering a state of suspended metabolism called anhydrobiosis. This unique ability is based on the specific molecular machinery involving a synthesis of non-reducing sugar trehalose and a variety of protective proteins. Genes encoding these protective proteins are extensively duplicated in the P. vanderplanki genome and become hugely upregulated in response to desiccation. Some of these highly expressed genes encode substitutions of amino acids crucial for the function of corresponding proteins. An intriguing group of protective proteins in P. vanderplanki are thioredoxins (TRX). These antioxidant proteins are important for P. vanderplanki anhydrobiosis since desiccation is tightly related to the elevated production of free radicals and oxidative damage. The TRX set is unprecedentedly expanded in the P. vanderplanki genome up to 25 TRX genes. Genomes of congeneric midge Polipedilum nubifer, Apis mellifera, Drosophila melanogaster, and Anopheles gambiae encode only 3–7 TRX genes. Moreover, all three P. vanderplanki thioredoxin genes most expressed at 24 h of P. vanderplanki larva desiccation encode for proteins missing the typical CxxC motif.

Regulating gonad inhibition and vitellogenin/vitellin induction in Penaeus monodon using mature GIH fusion protein and polyclonal antisera

Gonad inhibiting hormone (GIH), type II class of the CHH family neuropeptides, is released by the neurohaemal XO-SG complex of the eyestalk. The inhibitory function of GIH has a pivotal role in gonad development and reproduction. In this study, we report the expression and production of a thioredoxin-fused mature GIH protein (mf-PmGIH) of Penaeus monodon in a bacterial system and its use as antigen to raise polyclonal antiserum (anti-mf-PmGIH). The mature GIH gene of 237bp that codes for 79 amino acids, was cloned into the Escherichia coli thioredoxin gene fusion expression system. The expression vector construct (mf-PmGIH+pEt32a+) upon induction produced 32.16kDa mature GIH fusion protein (mf-PmGIH)·The purified fusion protein was used as exogenous GIH and as antigen to raise polyclonal antisera. The fusion protein when injected into juvenile shrimp significantly reduced vitellogenin/vitellin levels by 31.55% within 72h in comparison to the controls showing the gonad inhibiting property. Vitellogenin/vitellin levels were significantly induced by 74.10% within 6h when polyclonal antiserum (anti-mf-PmGIH - 1:500) was injected in P. monodon. Anti-mf-PmGIH immunolocalized GIH producing neurosecretory cells in the eyestalk of P. monodon. The present manuscript reports an innovative means of gonad inhibition and vitellogenin/vitellin induction with thioredoxin fused GIH and antisera developed.

Characteristics of Three Thioredoxin Genes and Their Role in Chilling Tolerance of Harvested Banana Fruit.

Thioredoxins (Trxs) are small proteins with a conserved redox active site WCGPC and are involved in a wide range of cellular redox processes. However, little information on the role of Trx in regulating low-temperature stress of harvested fruit is available. In this study, three full-length Trx cDNAs, designated MaTrx6, MaTrx9 and MaTrx12, were cloned from banana (Musa acuminata) fruit. Phylogenetic analysis and protein sequence alignments showed that MaTrx6 was grouped to h2 type with a typical active site of WCGPC, whereas MaTrx9 and MaTrx12 were assigned to atypical cys his-rich Trxs (ACHT) and h3 type with atypical active sites of GCAGC and WCSPC, respectively. Subcellular localization indicated that MaTrx6 and MaTrx12 were located in the plasma membrane and cytoplasm, respectively, whereas MaTrx9 showed a dual cytoplasmic and chloroplast localization. Application of ethylene induced chilling tolerance of harvested banana fruit, whereas 1-MCP, an inhibitor of ethylene perception, aggravated the development of chilling injury. RT-qPCR analysis showed that expression of MaTrx12 was up-regulated and down-regulated in ethylene- and 1-MCP-treated banana fruit at low temperature, respectively. Furthermore, heterologous expression of MaTrx12 in cytoplasmic Trx-deficient Saccharomyces cerevisiae strain increased the viability of the strain under H2O2. These results suggest that MaTrx12 plays an important role in the chilling tolerance of harvested banana fruit, possibly by regulating redox homeostasis.

Variability in gene content and expression of the thioredoxin system in Oenococcus oeni

The thioredoxin system protects against oxidative stress through the reversible oxidation of the thioredoxin active center dithiol to a disulphide. The genome of Oenococcus oeni PSU-1 contains three thioredoxin genes (trxA1, trxA2, trxA3), one thioredoxin reductase (trxB) and one ferredoxin reductase (fdr) which, until recently, was annotated as a second thioredoxin reductase. For the first time, the entire thioredoxin system in several O. oeni strains isolated from wine has been analysed. Comparisons at the DNA and protein levels have been undertaken between sequences from O. oeni and other genera and species, and the genera Leuconostoc and Lactobacillus were found to present the highest similarities. The gene most frequently absent from a collection of 34 strains and the sequences annotated in the NCBI database was trxA1. Moreover, phylogenetic analysis suggested that this gene was horizontally transferred from Lactobacillus to O. oeni. Strain-dependent expression profiles were determined in rich and in wine-like media. General over-expression was detected after inoculation into wine-like medium, with trxA3 being the most highly expressed gene. The increased transcriptional levels of the thioredoxin genes are indicative of the crucial role of this system in the O. oeni response to wine harsh conditions.

Cotesia vestalis parasitization suppresses expression of a Plutella xylostella thioredoxin.

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins involved in protecting organisms against toxic reactive oxygen species. In this study, a typical thioredoxin gene, PxTrx, was isolated from Plutella xylostella. The full-length cDNA sequence is composed of 959 bp containing a 321 bp open reading frame that encodes a predicted protein of 106 amino acids, a predicted molecular weight of 11.7 kDa and an isoelectric point of 5.03. PxTrx was mainly expressed in larval Malpighian tubules and the fat body. An enriched recombinant PxTrx had insulin disulphide reductase activity and stimulated Human Embryonic Kidney 293 (HEK293) cell proliferation. It also protected supercoiled DNA and living HEK293 cells from H2 O2 -induced damage. Parasitization by Cotesia vestalis and injections of 0.05 and 0.01 equivalents of C. vestalis Bracovirus (CvBv), the symbiotic virus carried by the parasitoid, led to down-regulation of PxTrx expression in host fat body. Taken together, our results indicate that PxTrx contributes to the maintenance of P. xylostella cellular haemostasis. Host fat body expression of PxTrx is strongly attenuated by parasitization and by injections of CvBv.

Thioredoxins are involved in the activation of the PMK1 MAP kinase pathway during appressorium penetration and invasive growth in Magnaporthe oryzae.

In Magnaporthe oryzae, the Mst11-Mst7-Pmk1 MAP kinase pathway is essential for appressorium formation and invasive growth. To determine their roles in Pmk1 activation and plant infection, we characterized the two thioredoxin genes, TRX1 and TRX2, in M. oryzae. Whereas the Δtrx1 mutants had no detectable phenotypes, deletion of TRX2 caused pleiotropic defects in growth, conidiation, light sensing, responses to stresses and plant infection progresses. The Δtrx1 Δtrx2 double mutant had more severe defects than the Δtrx2 mutant and was non-pathogenic in infection assays. The Δtrx2 and Δtrx1 Δtrx2 mutant rarely formed appressoria on hyphal tips and were defective in invasive growth after penetration. Pmk1 phosphorylation was barely detectable in the Δtrx2 and Δtrx1 Δtrx2 mutants. Deletion of TRX2 affected proper folding or intra-/inter-molecular interaction of Mst7 and expression of the dominant active MST7 allele partially rescued the defects of the Δtrx1 Δtrx2 mutant. Furthermore, Cys305 is important for Mst7 function and Trx2 directly interacts with Mst7 in co-IP assays. Our data indicated that thioredoxins play important roles in intra-cellular ROS signalling and pathogenesis in M. oryzae. As the predominant thioredoxin gene, TRX2 may regulate the activation of Pmk1 MAPK via its effects on Mst7.