Plant Thioredoxins Research Articles

Participation of the stress-responsive CDSP32 thioredoxin in the modulation of chloroplast ATP-synthase activity in Solanum tuberosum.

Plant thioredoxins (TRXs) are involved in numerous metabolic and signalling pathways, such as light-dependent regulation of photosynthesis. The atypical TRX CDSP32, chloroplastic drought-induced stress protein of 32 kDa, includes two TRX-fold domains and participates in responses to oxidative stress as an electron donor to other thiol reductases. Here, we further characterised potato lines modified for CDSP32 expression to clarify the physiological roles of the TRX. Upon high salt treatments, modified lines displayed changes in the abundance and redox status of CDSP32 antioxidant partners, and exhibited sensitivity to combined saline-alkaline stress. In non-stressed plants overexpressing CDSP32, a lower abundance of photosystem II subunits and ATP-synthase γ subunit was noticed. The CDSP32 co-suppressed line showed altered chlorophyll a fluorescence induction and impaired regulation of the transthylakoid membrane potential during dark/light and light/dark transitions. These data, in agreement with the previously reported interaction between CDSP32 and ATP-synthase γ subunit, suggest that CDSP32 affects the redox regulation of ATP-synthase activity. Consistently, modelling of protein complex 3-D structure indicates that CDSP32 could constitute a suitable partner of ATP-synthase γ subunit. We discuss the roles of the TRX in the regulation of both photosynthetic activity and enzymatic antioxidant network in relation with environmental conditions.

Battle for survival: the role of plant thioredoxin in the war against Barley stripe mosaic virus.

Battle for survival: the role of plant thioredoxin in the war against Barley stripe mosaic virus.

Thioredoxins: Emerging Players in the Regulation of Protein S-Nitrosation in Plants.

S-nitrosation has been recognized as an important mechanism of ubiquitous posttranslational modification of proteins on the basis of the attachment of the nitroso group to cysteine thiols. Reversible S-nitrosation, similarly to other redox-based modifications of protein thiols, has a profound effect on protein structure and activity and is considered as a convergence of signaling pathways of reactive nitrogen and oxygen species. This review summarizes the current knowledge on the emerging role of the thioredoxin-thioredoxin reductase (TRXR-TRX) system in protein denitrosation. Important advances have been recently achieved on plant thioredoxins (TRXs) and their properties, regulation, and functions in the control of protein S-nitrosation in plant root development, translation of photosynthetic light harvesting proteins, and immune responses. Future studies of plants with down- and upregulated TRXs together with the application of genomics and proteomics approaches will contribute to obtain new insights into plant S-nitrosothiol metabolism and its regulation.

High resolution crystal structure of NaTrxh from Nicotiana alata and its interaction with the S-RNase

Thioredoxins are regulatory proteins that reduce disulfide bonds on target proteins. NaTrxh, which belongs to the plant thioredoxin family h subgroup 2, interacts and reduces the S-RNase enhancing its ribonuclease activity seven-fold, resulting an essential protein for pollen rejection inNicotiana.Here, the crystal structure of NaTrxh at 1.7 Å by X-ray diffraction is reported. NaTrxh conserves the typical fold observed in other thioredoxins from prokaryotes and eukaryotes, but it contains extensions towards both N- and C-termini.The NaTrxh N-terminal extension participates in the reduction of S-RNase, and in the structure reported here, this is orientated towards the reactive site. The interaction between SF11-RNase and the NaTrxh N-terminal was simulated and the short-lived complex observed lasted for a tenth of ns. Moreover, we identified certain amino acids as SF11-RNase-E155 and NaTrxh-M104 as good candidates to contribute to the stability of the complex. Furthermore, we simulated the reduction of the C153-C186 SF11-RNase disulfide bond and observed subtle changes that affect the entire core, which might explain the increase in the ribonuclease activity of S-RNase when it is reduced by NaTrxh.

Lon domain-containing protein 1 represses thioredoxin y2 and regulates ROS levels in Arabidopsis chloroplasts.

Plant thioredoxins (Trxs) act as antioxidants and function as redox regulators in the chloroplast. Although the regulation of ROS in chloroplasts is well elucidated, the precise regulation mechanism of Trx remains unknown. Here, we characterize a novel chloroplast protein, Lon domain-containing protein 1 (LCP1), which contains only a Lon domain, the precise function of which is not known. We find that LCP1 interacts with Trx-y2 and represses its activity, and that knockdown (KD) of LCP1 causes anther indehiscence due to deficient lignin deposition. In addition, LCP1 KD plants show less ROS accumulation and lower expression of ROS-responsive marker genes than the wild-type plant. Taken together, we suggest that LCP1 directly regulates Trx-y2 and controls H2 O2 levels and, thereby, regulates lignin polymerization in the anther endothecium.

Molecular and Functional Characterization of a Rice Thioredoxin m Isoform and Its Interaction Proteins

Although subcellular localization and substrate specificity of thioredoxin isoforms have been characterized, there is little information on the specific functions of mtype plant thioredoxins or their interaction targets. Here, we describe the functional characterization of an Oryza sativa thioredoxin m (OsTrxm). We undertook yeast twohybrid screening using OsTrxm as a bait and found three interaction proteins, Pex14 and two Pex5 variants. Furthermore, two cysteines of OsTrxm were sufficient for the interaction between OsTrxm and these peroxisome proteins. To verify whether OsTrxm and the target proteins can be co-localized in vivo, we examined subcellular localization of OsTrxm-GFP and a peroxisomal marker RFP-SKL in Arabidopsis protoplast cells. Surprisingly, we detected OsTrxm localization in the cytosol and chloroplast. We confirmed these results by 2-D PAGE and Western blot analysis. Our results indicate that OsTrxm may play important roles in the cytoplasm for peroxisome biogenesis as well as in redox regulation of chloroplast proteins.

The Unprecedented Versatility of the Plant‎ Thioredoxin System.

Thioredoxins are ubiquitous enzymes catalyzing reversible disulfide-bond formation to regulate structure and function of many proteins in diverse organisms. In recent years, reverse genetics and biochemical approaches were used to resolve the functions, specificities, and interactions of the different thioredoxin isoforms and reduction systems in planta and revealed the most versatile thioredoxin system of all organisms. Here we review the emerging roles of the thioredoxin system, namely the integration of thylakoid energy transduction, metabolism, gene expression, growth, and development under fluctuating environmental conditions. We argue that these new developments help us to understand why plants organize such a divergent composition of thiol redox networks and provide insights into the regulatory hierarchy that operates between them.

Regulation of Differentiation of Nitrogen-Fixing Bacteria by Microsymbiont Targeting of Plant Thioredoxin s1

Legumes associate with rhizobia to form nitrogen (N2)-fixing nodules, which is important for plant fitness [1, 2]. Medicago truncatula controls the terminal differentiation of Sinorhizobium meliloti into N2-fixing bacteroids by producing defensin-likenodule-specific cysteine-rich peptides (NCRs) [3,4]. The redox state of NCRs influences some biological activities in free-living bacteria, but the relevance of redox regulation of NCRs in planta is unknown [5, 6], although redox regulation plays a crucial role in symbiotic nitrogen fixation [7, 8]. Two thioredoxins (Trx), Trx s1 and s2, define a new type of Trx and are expressed principally in nodules [9]. Here, we show that there are four Trx s genes, two of which, Trx s1 and s3, are induced in the nodule infection zone where bacterial differentiation occurs. Trx s1 is targeted to the symbiosomes, the N2-fixing organelles. Trx s1 interacted with NCR247 and NCR335 and increased the cytotoxic effect of NCR335 in S.meliloti. We show that Trx s silencing impairs bacteroid growth and endoreduplication, two features of terminal bacteroid differentiation, and that the ectopic expression of Trx s1 in S.meliloti partially complements the silencing phenotype. Thus, our findings show that Trx s1 is targeted to the bacterial endosymbiont, where it controls NCR activity and bacteroid terminal differentiation. Similarly, Trxs are critical for the activation of defensins produced against infectious microbes in mammalian hosts. Therefore, our results suggest the Trx-mediated regulation of host peptides as a conserved mechanism among symbiotic and pathogenic interactions.

Kinetic analysis of the interactions between plant thioredoxin and target proteins.

Thioredoxin is a critical protein that mediates the transfer of reducing equivalents in vivo and regulates redox sensitive enzymes in several cases. In addition, thioredoxin provides reducing equivalents to oxidoreductases such as peroxiredoxin. Through a dithiol–disulfide exchange reaction, the reduced form of thioredoxin preferentially interacts with the oxidized forms of targets, which are immediately released after this reaction is complete. In order to more thoroughly characterize these interactions between thioredoxin and its target proteins, a mutant version of thioredoxin that lacked the second cysteine was synthesized and interactions were monitored by surface plasmon resonance. The binding rates of thioredoxin to its targets were very different depending on the use of reducing equivalents by the targets: the enzymes whose activity was controlled by reduction or oxidation of a cysteine pair(s) in the molecule and the enzymes that used reducing equivalents provided by thioredoxin for their catalysis. In addition, thioredoxin revealed a stronger preference for an oxidized target. These results explain the reason for selective association of thioredoxin with oxidized targets for reduction, whereas immediate dissociation from a reduced target when the dithiol–disulfide exchange reaction is complete.

Role for Plant Thioredoxin in Cd2+ Chelation

Despite the critical role of thioredoxin (Trx) in redox regulation, little is known of the function in seed germination, especially in the presence of heavy metal. Plant redox reactions are principally studied on adult plant organs and there is little information on the onset of redox regulation during seed germination. Regulation of the antioxidant defense system during seed germination during the transition from heterotrophic growth to photoautotrophic metabolism in seedlings was examined in pea (Pisum sativum L.) in the presence of cadmium (Cd). Properties of the Cd2+-Trx interaction on the Trx from pea were examined to determine whether its binding affected Trx activity. When incubated with Cd in vitro the disulfide reductase activity of Trx o was inhibited. Disruption of the vicinal dithiol on Trx via oxidation to the disulfide (-S-S-) bridge protected against Cd and is consistent with a dithiol chelation mechanism. Thioredoxins are involved in defense and detoxification, antioxidant activity, and germination. Quantification of these proteins provides new insight for understanding the molecular bases of responses to heavy metal in seed during germination.

Single cystathionine β-synthase domain-containing proteins modulate development by regulating the thioredoxin system in Arabidopsis.

Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine β-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain-containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H(2)O(2) levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating photosynthesis-related [corrected] enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell.

Identification of uricase as a potential target of plant thioredoxin: Implication in the regulation of nodule development

During symbiotic nodule development in legume roots, early signaling events between host and rhizobia serve critical determinants for the proper onset of nodule morphogenesis, nitrogen fixation, and assimilation. Previously we isolated thioredoxin from soybean nodules as one of differentially expressed genes during nodulation and noted its positive role in nitrogen fixation. To identify the target proteins of thioredoxin in nodules, we used thioredoxin affinity chromatography followed by mass spectrometry. Nodulin-35, a subunit of uricase, was found to be a target of thioredoxin. Their interaction was confirmed by pull-down assay and by bimolecular fluorescent complementation. With an increased uricase activity observed also in the presence of thioredoxin, these results appear to implicate a novel role of thioredoxin in the regulation of enzyme activities involved in nodule development and nitrogen fixation.

Plant Thioredoxin CDSP32 Regenerates 1-Cys Methionine Sulfoxide Reductase B Activity through the Direct Reduction of Sulfenic Acid

Thioredoxins (Trxs) are ubiquitous enzymes catalyzing the reduction of disulfide bonds, thanks to a CXXC active site. Among their substrates, 2-Cys methionine sulfoxide reductases B (2-Cys MSRBs) reduce the R diastereoisomer of methionine sulfoxide (MetSO) and possess two redox-active Cys as follows: a catalytic Cys reducing MetSO and a resolving one, involved in disulfide bridge formation. The other MSRB type, 1-Cys MSRBs, possesses only the catalytic Cys, and their regeneration mechanisms by Trxs remain unclear. The plant plastidial Trx CDSP32 is able to provide 1-Cys MSRB with electrons. CDSP32 includes two Trx modules with one potential active site (219)CGPC(222) and three extra Cys. Here, we investigated the redox properties of recombinant Arabidopsis CDSP32 and delineated the biochemical mechanisms of MSRB regeneration by CDSP32. Free thiol titration and 4-acetamido-4'-maleimidyldistilbene-2,2'-disulfonic acid alkylation assays indicated that the Trx possesses only two redox-active Cys, very likely the Cys(219) and Cys(222). Protein electrophoresis analyses coupled to mass spectrometry revealed that CDSP32 forms a heterodimeric complex with MSRB1 via reduction of the sulfenic acid formed on MSRB1 catalytic Cys after MetSO reduction. MSR activity assays using variable CDSP32 amounts revealed that MSRB1 reduction proceeds with a 1:1 stoichiometry, and redox titrations indicated that CDSP32 and MSRB1 possess midpoints potentials of -337 and -328 mV at pH 7.9, respectively, indicating that regeneration of MSRB1 activity by the Trx through sulfenic acid reduction is thermodynamically feasible in physiological conditions.

Evidence of non-functional redundancy between two pea h-type thioredoxins by specificity and stability studies

The largest group of plant thioredoxins (TRXs) consists of the so-called h-type; their great number raises questions about their specific or redundant roles in plant cells. Pisum sativum thioredoxin h1 (PsTRX h1) and Pisum sativum thioredoxin h2 (PsTRX h2) are both h-type TRXs from pea ( Pisum sativum) previously identified and biochemically characterized. While both are involved in redox regulation and show a high-sequence identity (60%), they display different behavior during in vitro and in vivo assays. In this work, we show that these two proteins display different specificity in the capturing of protein targets in vitro, by the use of a new stringent method. PsTRX h2 interacted with classical antioxidant proteins, whereas PsTRX h1 showed a completely different pattern of targeted proteins, and was able to capture a transcription factor. We also showed that the two proteins display very different thermal and chemical stabilities. We suggest that the differences in thermal and chemical stability point to a distinct and characteristic pattern of protein specificity.

A novel extended family of stromal thioredoxins

Thioredoxins play key regulatory roles in chloroplasts by linking photosynthetic light reactions to a series of plastid functions. In addition to the established groups of thioredoxins, f, m, x, and y, novel plant thioredoxins were also considered to include WCRKC motif proteins, CDSP32, the APR proteins, the lilium proteins and HCF164. Despite their important roles, the subcellular locations of many novel thioredoxins has remained unknown. Here, we report a study of their subcellular location using the cDNA clone resources of TAIR. In addition to filling all gaps in the subcellular map of the established chloroplast thioredoxins f, m, x and y, we show that the members of the WCRKC family are targeted to the stroma and provide evidence for a stromal location of the lilium proteins. The combined data from this and related studies indicate a consistent stromal location of the known Arabidopsis chloroplast thioredoxins except for thylakoid-bound HCF164.

Thioredoxin h System and Wheat Seed Quality

ABSTRACTThioredoxin is one of the key systems controlling cellular redox balance in all living organisms. Plant thioredoxins are a diverse multigene family divided into two systems, the chloroplastic and the cytoplasmic systems, which are distinguishable by the electron donor and by the enzyme that catalyses thioredoxin reduction. In cereal seed, the thioredoxin (Trx) h system acts in the developing phase, controlling the delivery of compounds during seed filling. Early in the development of the imbibed seed, it promotes the mobilization of storage nitrogen and carbon in the endosperm by inhibiting the inactivators of amylolytic enzyme and by activating a specific serine protease, thiocalsin. During seed maturation and germination, the Trx h system controls oxidative stress in the living tissues, specifically in the scutellum and the aleurone layer, where it accumulates in the nucleus. The overexpression and the suppression of Trx confirm these features and constitute a powerful tool to manage seed quality, due to the large number of Trx h isoforms and to the specific nature of some of them.

Localization in roots and flowers of pea chloroplastic thioredoxin f and thioredoxin m proteins reveals new roles in nonphotosynthetic organs.

Plant thioredoxins (TRXs) are involved in redox regulation of a wide variety processes and usually exhibit organ specificity. We report strong evidence that chloroplastic TRXs are localized in heterotrophic tissues and suggest some ways in which they might participate in several metabolic and developmental processes. The promoter regions of the chloroplastic f and m1 TRX genes were isolated from a pea (Pisum sativum) plant genomic bank. Histochemical staining for beta-glucuronidase (GUS) in transgenic homozygous Arabidopsis (Arabidopsis thaliana) plants showed preferential expression of the 444-bp PsTRXf1 promoter in early seedlings, stems, leaves, and roots, as well as in flowers, stigma, pollen grains, and filaments. GUS activity under the control of the 1,874-bp PsTRXm1 promoter was restricted to the leaves, roots, seeds, and flowers. To gain insight into the translational regulation of these genes, a series of deletions of 5' elements in both TRX promoters were analyzed. The results revealed that a 126-bp construct of the PsTRXf2 promoter was unable to reproduce the expression pattern observed with the full promoter. The differences in expression and tissue specificity between PsTRXm1 and the deleted promoters PsTRXm2 and PsTRXm3 suggest the existence of upstream positive or negative regulatory regions that affect tissue specificity, sucrose metabolism, and light regulation. PsTRXm1 expression is finely regulated by light and possibly by other metabolic factors. In situ hybridization experiments confirmed new localizations of these chloroplastic TRX transcripts in vascular tissues and flowers, and therefore suggest possible new functions in heterotrophic tissues related to cell division, germination, and plant reproduction.

Thioredoxin and Redox Control within the New Concept of Oxidative Signaling

During the last decade, plant thioredoxins (TRX) h-type have been shown to be implicated in several new roles like the protection against the oxidative stress by their ability to reduce some antioxidant proteins as peroxiredoxins (PRX) or methionine-sulphoxide-reductases (MSR). However, the concept of the oxidative stress is changing and this fact raises the question of the TRX roles in this new context. In the January issue of Plant Physiology, we have presented two TRXsh from Pisum sativum differently involved in the control of the redox status. PsTRXh1 is an h-type TRX that acts by reducing classical antioxidant proteins. PsTRXh2 seems to be also involved in redox control, however it could act contrary to its counterpart h1. Both proteins may play antagonistic roles in pea in order to lead a better control of the redox status.

Thioredoxinh5 Is Required for Victorin Sensitivity Mediated by a CC-NBS-LRR Gene inArabidopsis

The fungus Cochliobolus victoriae causes Victoria blight of oats (Avena sativa) and is pathogenic due to its production of victorin, which induces programmed cell death in sensitive plants. Victorin sensitivity has been identified in Arabidopsis thaliana and is conferred by the dominant gene LOCUS ORCHESTRATING VICTORIN EFFECTS1 (LOV1), which encodes a coiled-coil-nucleotide binding site-leucine-rich repeat protein. We isolated 63 victorin-insensitive mutants, including 59 lov1 mutants and four locus of insensitivity to victorin1 (liv1) mutants. The LIV1 gene encodes thioredoxin h5 (ATTRX5), a member of a large family of disulfide oxidoreductases. To date, very few plant thioredoxins have been assigned specific, nonredundant functions. We found that the victorin response was highly specific to ATTRX5, as the closely related ATTRX3 could only partially compensate for loss of ATTRX5, even when overexpressed. We also created chimeric ATTRX5/ATTRX3 proteins, which identified the central portion of the protein as important for conferring specificity to ATTRX5. Furthermore, we found that ATTRX5, but not ATTRX3, is highly induced in sensitive Arabidopsis following victorin treatment. Finally, we determined that only the first of the two active-site Cys residues in ATTRX5 is required for the response to victorin, suggesting that ATTRX5 function in the victorin pathway involves an atypical mechanism of action.

Plant thioredoxins are key actors in the oxidative stress response

Thioredoxins are ubiquitous disulfide reductases that regulate the redox status of target proteins. Although plant thioredoxins display a striking diversity not found in other organisms, many of their physiological roles have yet to be determined. Based on recent publications investigating thioredoxin targets and genetically modified plants, thioredoxins appear to play a fundamental role in plant tolerance of oxidative stress. They are involved in oxidative damage avoidance by supplying reducing power to reductases detoxifying lipid hydroperoxides or repairing oxidized proteins. Furthermore, other lines of evidence indicate that thioredoxins could act as regulators of scavenging mechanisms and as components of signalling pathways in the plant antioxidant network.