Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development

doi:10.1105/tpc.107.050849

Journal List > Plant Cell > v.19(6); Jun 2007

Plant Cell. 2007 June; 19(6): 1851–1865.

doi: 10.1105/tpc.107.050849.

PMCID: PMC1955716

Inactivation of Thioredoxin Reductases Reveals a Complex Interplay between Thioredoxin and Glutathione Pathways in Arabidopsis Development^[W]

Jean-Philippe Reichheld,^a¹ Mehdi Khafif,^a² Christophe Riondet,^a Michel Droux,^b Géraldine Bonnard,^c and Yves Meyer^a

^aLaboratoire Génome et Développement des Plantes, Université de Perpignan, Unité Mixte de Recherche, Centre National de la Recherche Scientifique 5096, 66860 Perpignan, France

^bLaboratoire Mixte Centre National de la Recherche Scientifique/Bayer CropScience Unité Mixte de Recherche 5240, 69263 Lyon Cedex 9, France

^cInstitut de Biologie Moléculaire des Plantes, Unité Propre de Recherche, Centre National de la Recherche Scientifique 2357, Université Louis Pasteur, 67084 Strasbourg, France

¹To whom correspondence should be addressed. E-mail jpr/at/univ-perp.fr; fax 33-4-68-66-84-99.

²Current address: Institut des Sciences du Végétal, Unité Propre de Recherche Centre National de la Recherche Scientifique 2355, Bat 23, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.

Received February 6, 2007; Revised April 30, 2007; Accepted May 29, 2007.

This article has been cited by other articles in PMC.

Abstract

NADPH-dependent thioredoxin reductases (NTRs) are key regulatory enzymes determining the redox state of the thioredoxin system. The Arabidopsis thaliana genome has two genes coding for NTRs (NTRA and NTRB), both of which encode mitochondrial and cytosolic isoforms. Surprisingly, plants of the ntra ntrb knockout mutant are viable and fertile, although with a wrinkled seed phenotype, slower plant growth, and pollen with reduced fitness. Thus, in contrast with mammals, our data demonstrate that neither cytosolic nor mitochondrial NTRs are essential in plants. Nevertheless, in the double mutant, the cytosolic thioredoxin h3 is only partially oxidized, suggesting an alternative mechanism for thioredoxin reduction. Plant growth in ntra ntrb plants is hypersensitive to buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis, and thioredoxin h3 is totally oxidized under this treatment. Interestingly, this BSO-mediated growth arrest is fully reversible, suggesting that BSO induces a growth arrest signal but not a toxic accumulation of activated oxygen species. Moreover, crossing ntra ntrb with rootmeristemless1, a mutant blocked in root growth due to strongly reduced glutathione synthesis, led to complete inhibition of both shoot and root growth, indicating that either the NTR or the glutathione pathway is required for postembryonic activity in the apical meristem.

INTRODUCTION

Thiol-disulfide oxidoreductases constitute a large protein family characterized by the typical active site CxxC that performs the oxidation, reduction, or isomerization of disulfide bonds of target proteins. Thioredoxins (TRXs) in this family are characterized by the canonical active site WCG/PPC, with a low redox potential that confers strong reductive properties. In their reduced state, thioredoxins are able to provide reducing power to numerous target proteins like peroxidases or reductases (Carmel-Harel and Storz, 2000; Nordberg and Arner, 2001). Moreover, they also play signaling roles through structural modifications of target proteins, such as several Calvin cycle enzymes of plant chloroplasts (Ruelland and Miginiac-Maslow, 1999). Subsequently, the oxidized thioredoxins are reduced by thioredoxin reductase, together forming the so-called NADPH-dependent thioredoxin system (NTS) (Holmgren, 1985).

In mammals, functional NTSs have been found in the cytosol and mitochondria, both encoded by single members. The central role of both cytosolic and mitochondrial NTSs was exemplified by genetic approaches. In mouse, disruption of the unique gene encoding the cytosolic TRX leads to early embryonic lethality (Matsui et al., 1996). Similarly, disruption of the gene encoding the mitochondrial NADPH-dependent thioredoxin reductase (NTR) impairs embryo development because of severe growth retardation or cardiac development (Jakupoglu et al., 2005). NTSs are involved in many cellular processes (Arner and Holmgren, 2000), exerting cytokine-like functions (Nishinaka et al., 2002), modulating the activity of redox-regulated transcription factors such as NF-kB (Qin et al., 1995) and AP-1 (Karimpour et al., 2002), mediating peroxiredoxin antioxidant properties (Verdoucq et al., 1999), and being putatively involved in DNA synthesis through the reduction of ribonucleotide reductase (Koc et al., 2006).

Plants are distinguishable from other organisms by their very complex thioredoxin system, as revealed by sequencing of the Arabidopsis thaliana genome (Arabidopsis Genome Initiative, 2000). Approximately 40 genes coding for TRX and TRX-related proteins have been identified. Among them, at least 11 belong to the cytosolic TRXh group, but additional TRX-like proteins are predicted to have a cytosolic localization (Meyer et al., 2006). A functional mitochondrial NTS system has been identified in Arabidopsis (Laloi et al., 2001). Moreover, the chloroplastic TRX system has been studied extensively in plants (Schürmann and Jacquot, 2000). Typically, chloroplastic thioredoxins (TRXm, f, x, and y) are reduced in the light by a ferredoxin-dependent heterodimeric thioredoxin reductase (FTR) homolog of the cyanobacterial FTRs (Dai et al., 2000). Reverse genetic studies suggest an antioxidant role for the FTR pathway (Keryer et al., 2004). Recently, an atypical bidomain NTR-TRX–like thioredoxin reductase (NTRC) was proposed to relay the FTR detoxification system during the night (Serrato et al., 2004; Perez-Ruiz et al., 2006).

Genetic approaches aiming to identify functions of cytosolic thioredoxins in knockout plants have been largely limited by the absence of phenotypes of single mutants, presumably due to functional redundancies among members of the multigene families of TRX. In one report, virus-induced gene silencing of the cytosolic TRX-like protein CITRX convincingly demonstrated its implication in the Cf9/Avr9-triggered hypersensitive response (Rivas et al., 2004). The alternative NADPH-dependent redox pathway, which implicates glutaredoxins, is a potential source of redundancy. Glutaredoxins belong to the TRX superfamily according to their structure and related disulfide reductase activity. They mainly differ from the latter by the fact that they use GSH as an electron donor instead of thioredoxin reductase (Rouhier et al., 2004). Subsequently, GSSG is reduced by glutathione reductase (GR), an NADPH-dependent flavoprotein related to NTR. Hereafter, this system will be called the NADPH-dependent glutaredoxin system (NGS). These two systems are known to present some functional redundancies and to share common target proteins in Escherichia coli, Saccharomyces cerevisiae, and plants (Carmel-Harel and Storz, 2000; Rouhier et al., 2001). In budding yeast, a single TRX or GRX gene is necessary to maintain cell viability, emphasizing redundancy between both redoxins (Draculic et al., 2000).

In order to overcome the redundancy within redox pathways and to gain more insight on the functions of NTS in plants, we inactivated NTRs, the main reductor of TRX in Arabidopsis. In previous studies, we have shown that both NTRA and NTRB genes encode cytosolic and mitochondrial isoforms (Reichheld et al., 2005), suggesting that the double ntra ntrb mutant inactivates both cytosolic and mitochondrial NTSs. We conclude from the characterization of this double mutant that the NTSs may play important roles in several plant development programs, including pollen fitness, seed development, and cell proliferation. Differential complementation experiments indicate that phenotypes of the diploid phase of ntra ntrb are due to inactivation of the cytosolic NTS system. Furthermore, the triple ntra ntrb rootmeristemless1 (rml1) mutant provides genetic evidence in plants of the conjunction of the NTS and glutathione pathways in setting up postembryonic meristematic activities.

RESULTS

Redundancy between NTRA and NTRB

In order to characterize the functions of NTR in Arabidopsis, we isolated T-DNA insertion mutants from the SALK library (Alonso et al., 2003) in each of the NTRA (SALK_539152) and NTRB (SALK_545978) genes. We previously established that this insertion accurately inactivates each gene (Reichheld et al., 2005). However, the corresponding ntra and ntrb mutant plants failed to reveal any phenotype. This suggested that the NTRA and NTRB proteins may share redundant functions. To gain more insight on the expression of the two genes in plants, we studied the steady state levels of the two NTR transcripts in different plant organs by RT-PCR. Both genes were ubiquitously expressed in all studied organs and showed similar expression profiles (Figure 1A). To gain further information about the individual expression of NTRA and NTRB genes during plant development, we studied their expression patterns using promoter:β-glucuronidase (GUS) fusion genes (Figures 1B to 1M). Similar GUS staining was observed in Pro_NTRA:GUS (Figures 1B to 1G) and Pro_NTRB:GUS (Figures 1H to 1M) plants. Expression of GUS genes was high in the shoot apex and young emerging leaves. Vascular tissues of the whole plant were stained as well as flower buds and developing flowers. Remarkably, pollen grains as well as germinating pollen tubes were highly stained in Pro_NTRA:GUS plants. This staining was also found in Pro_NTRB:GUS plants, although at a lower level. The root apex was stained in both constructs, with staining in Pro_NTRA:GUS restricted to the columella and Pro_NTRB:GUS plants showing faint staining of the central zone of the shoot apex. In both types of plants, no staining was detected during early embryo maturation. However, mature embryos and germinating plants were stained in both constructs.

Figure 1.

Expression of NTRA and NTRB Genes.

The ntra ntrb Double Mutant Is Viable but the Pollen Has Reduced Fitness

In order to cope with the redundancy between NTRA and NTRB, homozygous ntra and ntrb plants were crossed and plants from the progeny were screened to find homozygous ntra ntrb plants. In a first screening of 80 plants of the F2 generation, no double ntra/ntra ntrb/ntrb homozygous plant was found. To increase the probability of finding double homozygous plants, a plant harboring the ntra/ntra NTRB/ntrb genotype was selected, and 60 plants of its progeny were genotyped. In this population, only three plants (5%) having a ntra/ntra ntrb/ntrb homozygous genotype were found, whereas other plants were ntra/ntra NTRB/NTRB (42%) or ntra/ntra NTRB/ntrb (53%). This suggested that the ntra ntrb mutations do not follow Mendelian segregation (χ² = 9.6, P < 0.01). We reasoned that this non-Mendelian segregation could have a gametophytic origin. A female gametophytic effect is usually visualized by aborted seeds in siliques. We did not observe aborted seeds in the ntra/ntra NTRB/ntrb siliques (data not shown). Alternatively, a male gametophytic origin would be in agreement with GUS staining found in pollen grains and germinating pollen. Anthers of ntra/ntra NTRB/ntrb plants did not reveal reduced numbers or lethality of pollen grains by Alexander staining (data not shown).

To further study loss of fertility in mutant pollen, we performed pollen competition experiments between mutant and wild-type pollen (Table 1). Pistils of ntra ntrb plants were pollinated by pollen of ecotype Columbia (Col-0), ntra ntrb, or ntra/ntra NTRB/ntrb plants, and percentages of homozygous versus heterozygous plants from the progeny were determined. This discrimination was based on the higher sensitivity of homozygous ntra/ntra ntrb/ntrb plants to the glutathione inhibitor buthionine sulfoximine (BSO) compared with NTRA/ntra NTRB/ntrb or ntra/ntra NTRB/ntrb heterozygotes (see below). As expected, when ntra ntrb pistils were pollinated with pollen of Col-0 or ntra ntrb plants, 100% heterozygous or homozygous seeds, respectively, were found in the progeny (Table 1), showing that no residual self-pollinization of the ntra ntrb pistils occurred during the experiment. A pollen competition experiment was performed by pollination of ntra ntrb pistils with pollen of ntra/ntra NTRB/ntrb plants. If there were no decrease in the fertility of ntra ntrb pollen compared with ntra NTRB pollen, one could expect to find a ratio of 50% heterozygous ntra/ntra NTRB/ntrb to 50% homozygous ntra/ntra ntrb/ntrb seeds. Only 25% homozygous ntra/ntra ntrb/ntrb plants were actually obtained, which clearly diverges from what was expected (χ² = 35.5, P < 0.001). This indicates that ntra ntrb pollen fertilizes ovules less efficiently than ntra NTRB pollen.

Table 1.

Pollen Competition Experiments

Inactivation of NTRs Leads to Slowed Plant Growth, Modified Seed Shape, and High Accumulation of Anthocyanins, but the Mutant Is Fully Fertile and Seedlings Are Not Hypersensitive to Oxidant Stresses

The progeny of ntra ntrb homozygous plants were further analyzed. One hundred percent of these plants looked significantly smaller than wild-type plants or single homozygous ntra or ntrb mutant plants (Figures 2B and 2C). To verify that both NTR genes are inactivated in these plants, steady state levels of NTRA and NTRB proteins were measured by protein gel blot. As described previously, anti-NTRB antibodies were able to recognize both NTRA and NTRB proteins. Compared with the single ntra or ntrb homozygous mutant, in which NTRB or NTRA protein, respectively, was detected, accumulation of both NTRA and NTRB proteins was completely abolished in ntra ntrb (Figure 2A).

Figure 2.

Slower Growth of the ntra ntrb Mutant Is Complemented by the NTRB Gene.

In order to determine that the phenotypic characteristics were linked to the inactivation of NTR genes, the ntra ntrb mutant was complemented by a 3.6-kb genomic fragment containing the NTRB gene. Among 15 transformed plants containing the transgene, three were characterized further. In all three lines, the NTRB protein was recovered, showing that the transgene is functional (Figure 2A). All three complemented plants recovered a growth rate comparable to that of wild-type plants, showing that at least the NTRB protein is sufficient to confer a normal growth rate to the plant (Figure 2B). This is consistent with the absence of phenotype in the ntra single mutant. Interestingly, the C11 line that expresses the transgene at a lower level than Col-0 recovers the same growth rate as Col-0, C1, and C10, indicating that the NTR level is not a limiting factor for plant growth in the wild type, at least under standard conditions.

To study in more detail the impact of NTR inactivation on plant growth, we compared the development of the ntra ntrb mutant with that of wild-type and complemented lines. Germination kinetics and rate were not affected in the mutant, but leaf emergence (Figure 2C) and bolting were delayed slightly, resulting in delayed growth of the inflorescence stems (see Supplemental Figure 1 online).

Smaller leaves can result from a cell division and/or cell elongation defect. To clarify this point, we compared the density of abaxial epidermal cells in the third leaf of the mutant with that of wild-type plants. The cell density was found to be reduced, indicating that the inactivation of NTRs more likely affects cell division rate rather than cell elongation (Figure 2F). Moreover, cross sections of leaves indicate that the architecture of the leaf was not perturbed in the ntra ntrb mutant (Figures 2D and 2E).

The impact of NTR inactivation in roots was also studied. Daily root growth was measured in the ntra ntrb mutant and compared with that in Col-0 and complemented lines (Figure 3A). Root growth was reproducibly reduced by 30 to 35% in the ntra ntrb mutant compared with wild-type plants. In three complemented lines (C1, C10, and C11), root growth recovered almost the same level as the wild-type ecotype. However, single mutations of NTRA or NTRB did not influence root growth (data not shown). This indicates that NTRs participate in root growth and that at least one NTR is necessary to ensure proper root growth. The lower root growth rate in the ntra ntrb mutant is correlated with some modifications observed in the primary root tip of the mutant, in which bigger cells were observed (Figures 3B and 3C).

Figure 3.

Slower Root Growth of the ntra ntrb Mutant.

Phenotypic modifications of the ntra ntrb mutant were further characterized. Mutant seeds were slightly wrinkled (Figure 4A). Wrinkled seeds are often associated with perturbation of the accumulation of storage components (i.e., proteins and lipids). Storage proteins and lipids were quantified in ntra ntrb mutant seeds, but neither the rate nor the quality of storage proteins and lipids was modified compared with wild-type seeds (data not shown). Further studies should be performed in order to understand the origin of this phenotype. As a first indication, germinated seeds harbor ovoid cotyledons in which the vasculature is simpler than in wild-type plantlets (Figure 4B). This suggests that the thinner shape of the ntra ntrb cotyledons could account for the wrinkled seed phenotype. In some case, monocotyledonous, tricotyledonous, or even quadricotyledonous plants were observed (3 to 5% plants), suggesting that perturbations sometimes occur at early stages of embryonic development (Figure 4C). Visual examination of the mutant shows that it is clearly darker than the wild type. This is due to anthocyanins, which are ~3 to 10 times more abundant in the mutant than in the wild type (data not shown). The ntra ntrb plant is fully fertile, suggesting that reduced fitness of the mutant pollen is disadvantageous in competition with the wild type but that fertility of the mutant plant after self-pollination is not impaired.

Figure 4.

Wrinkled Seeds of the ntra ntrb Mutant.

As the NTS system was shown to be implicated in a stress response pathway, we subjected ntra ntrb plants to several abiotic stress conditions. Plants growing in vitro were transferred to medium containing increasing concentrations of reagents that generate cellular oxidative stress: redox quinone menadione, H₂O₂, and the heavy metals CdCl₂ and CuCl₂. Plant growth of the ntra ntrb plants was compared with that in wild-type plants. Otherwise, seeds were germinated directly in the same medium. ntra ntrb plants growing on soil were also subjected to high-light conditions (>10,000 lux) or challenged with virulent or avirulent stains of Pseudomonas syringae (in collaboration with D. Roby, Institut National de la Recherche Agronomique, Castanet-Tolosan). Surprisingly, mutant plants did not show significantly modified sensitivity to any of the conditions studied (data not shown).

Inactivation of NTR Is Compensated by Glutathione

The rather limited effects of NTR inactivation might be explained by redundant function with other redox pathways involving ascorbate or glutathione. We used the well-known ascorbate biosynthesis inhibitor lycorine to alter the pool of ascorbate. This was shown to drop to 15% under a 50 μM lycorine treatment and to affect plant growth. However, both ntra ntrb and wild-type plants showed similar growth kinetics when treated with different concentrations of lycorine (0 to 100 μM) (data not shown).

We next studied the impact of lowering the level of glutathione on the growth of the ntra ntrb mutant. We germinated wild-type and ntra ntrb plants on medium containing BSO. BSO is a nontoxic and highly specific inhibitor of the first enzyme of GSH biosynthesis, γ-glutamyl cysteine synthetase (γ-ECS), and its application results in the depletion of cellular GSH. Depletion of glutathione was shown to affect specifically root meristematic activity (Sanchez-Fernandez et al., 1997; Vernoux et al., 2000). Therefore, we compared the root growth of wild-type and ntr mutants subjected to BSO treatment. A low concentration of BSO (0.5 mM) did not affect the root growth of wild-type plants (cf. the growth of Col-0 plants in Figure 5B with that in Figure 3A). However, the same concentration of BSO drastically inhibited root growth of the ntra ntrb mutant, which stopped completely at 4 d after germination. In order to confirm that the hypersensitivity of the ntra ntrb mutant to BSO is linked to the inactivation of NTRs, complemented plants were subjected to the same BSO treatment. Root growth was hardly affected in these lines (Figure 5B). Moreover, single ntra and ntrb mutants were also not affected by BSO (data not shown).

Figure 5.

Hypersensitivity of the ntra ntrb Mutant to Glutathione Biosynthesis Inhibition by BSO.

These data demonstrate that glutathione is able to complement the absence of NTRs in the ntra ntrb mutant plants. Interestingly, when the BSO-treated mutants are transferred to a medium that does not contain BSO, root growth rapidly recovers to levels similar to those of wild-type plants (Figure 5B). This rapid reversibility indicates that the root growth inhibition observed in the ntra ntrb mutant is not due to a toxic accumulation of activated oxygen species in roots but more likely to a transient arrest of plant growth. Moreover, shoot development is also inhibited in the mutant under this BSO treatment, indicating that the effect of BSO on plant growth is not restricted to root development (Figures 5A and 9E).

Figure 9.

Differential Complementation of the ntra ntrb Mutant by Cytoplasmic or Mitochondrial Isoforms of NTRB.

In order to further address the question of the role of glutathione in compensating the loss of NTRs, we measured the pools of glutathione in the ntr mutants. The levels of GSH and GSSG were both slightly lower in the ntra ntrb mutant compared with wild-type plants (−17% and −13%, respectively) (see Supplemental Table 1 online). Moreover, the GSH/GSH+GSSG redox ratio indicates that most glutathione is in the reduced form in both ntra ntrb and wild-type plants. These data indicate that inactivation of the NTRs does not strongly influence the pools of glutathione. As glutathione is considered the major cellular redox buffer, we can conclude that ntra ntrb plants are not suffering from modifications in their cellular redox state. The slightly lower level of total glutathione in the ntra ntrb mutant may reflect the slower cell division rate occurring in the ntra ntrb plants.

Meristematic Activities Are Impaired in the ntra ntrb rml1 Mutant

In order to confirm genetically the role of glutathione in the ntra ntrb mutant, we crossed the ntra ntrb mutant with the rml1 mutant (Cheng et al., 1995). This mutation was shown to segregate as a single recessive allele. The mutant allele harbors a substitution mutation (D258N) in the first enzyme of glutathione biosynthesis, γ-ECS, resulting in an ~97% reduction of extractible GSH (Vernoux et al., 2000). This mutant is unable to establish an active postembryonic meristem in the root apex, due to abolished cell division in the root but not in the shoot. Although the shoot meristem is still active in rml1, the homozygous mutant is sterile. Therefore, a homozygous ntra ntrb mutant was crossed with rml1/+ heterozygous plants. Progeny of selected F2 plants were further analyzed (Table 2): 73.2% of the progeny of plants harboring the NTRA/NTRA NTRB/ntrb rml1/RML1 genotype show a wild-type phenotype, whereas 26.7% of the progeny show an rml1/rml1 phenotype (χ² = 0.31, P > 0.5), confirming that the rml1 mutation segregates as a single recessive allele in the studied genetic background. A total of 74.7% of the progeny of ntra/ntra ntrb/ntrb rml1/RML1 plants show a phenotype similar to ntra ntrb homozygous plants. However, 25.3% of the progeny show a much more perturbed development than rml1/rml1 homozygous plants: 14.3% of the seeds do not germinate, and the 11.0% that do germinate do not grow further. The approximate ratio (75/25; χ² = 0.011, P > 0.9) indicates Mendelian segregation of the rml1 allele and suggests that the most perturbed progeny are triple ntra ntrb rml1 homozygotes. Moreover, by genotyping some of the presumed triple homozygotes, we established that all of them are homozygous for the ntra ntrb and rml1 mutations. Phenotypically, it appears that the ntra ntrb rml1 plants do not show any shoot and root meristematic activity (Figure 6). This phenotype contrasts with that of the rml1 plants, in which root but not shoot meristem activity is affected, as shown by the emerging leaf primordia. Therefore, we conclude that NTR or glutathione is required to activate shoot postembryonic meristematic activities. Meristematic activities of most ntra ntrb rml1 plants are rescued when transferred to Murashige and Skoog (MS) medium containing GSH. This reversibility indicates that the triple ntra ntrb rml mutation is not lethal for the plant and does not alter the potential of the meristematic cells. The germination rate of the ntra ntrb rml1 seeds is also perturbed (Table 2), indicating that the NTS and glutathione pathways are involved in germination functions. This effect on germination was also partially rescued by GSH: 43.3% of the nongerminated ntra ntrb rml1 seeds were able to germinate when transferred to MS medium containing GSH.

Table 2.

Segregation of the rml1 Mutation on the ntra ntrb Background

Figure 6.

Phenotype of the ntra ntrb rml1 Mutant.

Alternative Role of NTR and Glutathione to Maintain the Redox State of TRXh

We next examined how the inactivation of NTRs affects the redox state of TRXs in planta. The cellular oxidation state was preserved by rapidly treating cells with trichloracetic acid, which protonates free thiol groups. Plant extracts were reacted with the thiol-specific reagent 4-acetamido-4′-maleimidyldistilbene-2,2′-disulfonic acid (AMS), followed by separation by SDS-PAGE. The redox state of the most abundant cytosolic TRXh3 protein was then determined by protein gel blot analysis (Figure 7A). The migration of all TRXh3 proteins from the wild type was decreased after treatment with AMS, indicating that the vast majority of TRXh3 is present in a reduced form in Col-0. The mutation of either ntra or ntrb does not perturb the redox state of TRXh3, indicating a compensation effect of NTRA and NTRB on the reduction of TRXh3 (data not shown). As might be expected, the loss of both NTRA and NTRB proteins shifted the TRXh3 balance to a more oxidized ratio, with approximately equal amounts of reduced and oxidized forms. However, the presence of high concentrations of reduced TRXh3 is somewhat surprising and suggests that another thioredoxin-reducing mechanism might exist to recycle the oxidized pool of TRXh3 in the absence of NTR.

Figure 7.

Redox State of Thioredoxin At TRXh3 in the ntra ntrb Mutant.

With respect to the role of glutathione in the development of the ntra ntrb mutant, we reasoned that GSH would also account for an alternative reduction of TRXh in the ntra ntrb mutant. We measured the amount and redox state of TRXh3 in ntra ntrb plants subjected to BSO treatment (Figure 7B). However, this treatment markedly increases the pool of TRXh3 protein in both Col-0 and ntra ntrb. Moreover, BSO (0.5 mM) has no effect on the redox state of TRXh3 in Col-0 plants but dramatically perturbs the redox state of TRXh3 in the ntra ntrb background, in which it is mostly oxidized. This indicates that decreasing the pool of GSH interferes with the reduction of TRXh3. ntra ntrb plants complemented with the NTRB gene show the same redox state of TRXh3 as wild-type plants, indicating that NTRB is sufficient to reduce TRXh3. In order to check whether this oxidation of TRXh3 is specific to BSO and not due to a general oxidation effect in plants, we subjected ntra ntrb plants grown in vitro to oxidative conditions triggered by increasing concentrations of H₂O₂ (1 to 10 mM) or menadione (10 to 100 μM). None of these conditions perturbed the redox state of TRXh3 (data not shown). Overall, these data indicate that the glutathione pathway is implicated in the alternative reduction of TRXh3 and that plants respond to perturbation of the GSH pool by BSO by increasing the pool of TRXh3.

Glutaredoxins but Not Glutathione Can Reduce Thioredoxins in Vitro

In order to decipher the mechanism of alternative reduction of TRXh3 by the glutathione pathway, we performed biochemical tests using the classical TRX-dependent insulin reduction test (Figure 8). As expected, the At TRXh3 protein is able to reduce the insulin-disulfide bridges using the thioredoxin reductase NTRA and NADPH as reducing power. However, it is poorly reduced by the E. coli GR or by the GSH/GR system, indicating that TRXh3 cannot be reduced directly by the GSH pool. However, the TRXh3-dependent insulin reduction was significantly increased by At GRX1 (At5g40370) or At GRX4 (At5g63030), indicating that the electron flux can be transferred to TRXhs via the NADPH/GR/GSH/GRX system.

Figure 8.

Biochemical Activity of At GRX on At TRXh3.

The Phenotype of the ntra ntrb Mutants in Their Diploid Phase Is Due to Inactivation of the Cytosolic NTR/TRXh System

The fact that both NTRA and NTRB genes code for mitochondrial and cytosolic isoforms raises the question of whether the phenotypes of ntra ntrb are due to inactivation of the cytosolic or the mitochondrial NTS system. In order to discriminate between these two different systems, we performed a complementation of the ntra ntrb mutant using constructs expressing either the mitochondrial or the cytosolic isoform of the NTRB cDNA (Figure 9A).

Mitochondrial and cytosolic fractions were prepared from Col-0 and ntra ntrb, as well as from NTRBc and NTRBm complemented plants. Protein gel blots were hybridized with anti-NTRB antibodies, which recognized both NTRA and NTRB proteins (Reichheld et al., 2005). In agreement with the expected molecular mass of the cytosolic isoform of NTRA and NTRB, a single band of ~35 kD was revealed in the cytosolic fractions of Col-0 and NTRBc plants. This band was more intense in NTRBc, suggesting that the NTRBc gene is overexpressed in this line. Surprisingly, a signal was also found in the mitochondrial fraction of NTRBc plants. This peptide migrates at the same level as the cytosolic isoform of NTRB. According to previous data, it is unlikely that this isoform is truly imported into mitochondria and more likely corresponds to proteins that were copurified with the mitochondrial fraction, possibly related to the high level of expression. A signal of ~37 kD was detected in the mitochondrial fractions of Col-0 and NTRBm. This band corresponds to the expected molecular mass of the mature mitochondrial NTRB isoform and suggests that the NTRBm protein was correctly targeted and matured.

Phenotypes of NTRBm and NTRBc plants were compared with those of Col-0 and ntra ntrb plants. NTRBc seeds recovered a shape similar to Col-0 seeds, while NTRBm plants were not complemented for the wrinkled phenotype (Figure 9B). NTRBc complemented plants recovered a growth rate similar to Col-0 plants compared with plants complemented with the NTRBm isoform (Figure 9C). Similarly, NTRBc plants recovered resistance to BSO, while NTRBm plants were as sensitive as the ntra ntrb mutant (Figure 9D). NTRBc plants present an anthocyanin content similar to that of Col-0, while NTRBm plants present high anthocyanin content similar to the ntra ntrb mutant (data not shown). These data clearly indicate that the somatic phenotypes of the ntra ntrb mutant are due to the inactivation of the cytosolic form of NTR.

DISCUSSION

NTRA and NTRB Have Redundant Functions and Are Not Essential for Plant Development

The ntra and ntrb single knockout mutants do not show any visible phenotypic modifications. This is consistent with the high similarity (95% amino acid identity) of the encoded proteins, the dual cytosolic and mitochondrial localization of the NTRs, and the almost identical expression pattern of both genes. It can be concluded that NTRA and NTRB proteins share almost redundant functions in plants. Nevertheless, the NTR genes derive from one duplication of a large chromosomal fragment in an ancestor of the Brassicaceae (Reichheld et al., 2005). A copy of most of their neighboring genes was lost during subsequent evolution, suggesting that some selective pressure occurs in favor of the maintenance of both NTR copies in the genome. Therefore, we cannot exclude the possibility that the distinct NTRA and NTRB may have some specific functions under particular environmental conditions.

The most obvious impact of the ntra ntrb mutation is its influence on plant growth. During leaf formation, a phase of cell division is followed by cell expansion and functional differentiation. The ntra ntrb mutation led to a significant reduction of the leaf area, which was partially compensated by increased cell size, indicating that the primary cause is decreased cell division during leaf formation. The role of NTR on cell division rate has been documented previously in yeast. Loss of thioredoxin reductase trr1 led to a slower growth rate (Trotter and Grant, 2003). Similarly, the double thioredoxin mutant trx1 trx2 harbors defects in cell cycle progression because of a longer S-phase. In vivo evidence shows that the trx1 trx2 mutant is not able to support the high rates of deoxynucleotide triphosphate synthesis required for efficient DNA replication, indicating that thioredoxin is the preferred physiological electron donor for ribonucleotide reductase in yeast (Koc et al., 2006). Although ribonucleotide reductase is one candidate explanation for the decreased growth rate in ntra ntrb, alternative targets must be considered. Mammalian phosphatases CDC25, direct activators of CDK–cyclin complexes, have been shown to be regulated in a TRX-dependent manner (Sohn and Rudolph, 2003). Furthermore, the role of glutathione on cell cycle progression has been largely documented in mammals and plants (Russo et al., 1995; Vernoux et al., 2000; Potters et al., 2004; reviewed in Maughan and Foyer, 2006). Whether both NTR and glutathione pathways could regulate a common cell cycle regulator will have to be determined.

The growth delay of the ntra ntrb mutant constitutes a means of counterselection of the ntra ntrb homozygous mutation, as these smaller plants should suffer from light limitation by shading from neighboring wild-type plants. The non-Mendelian transmission rate of the ntrb and wild-type alleles in the progeny of ntra/ntra NTRB/ntrb plants constitutes another type of counterselection. Male gametophytic origin is suggested by the pollen competition experiments and further sustained by both Pr NTRA:GUS and Pr NTRB:GUS plants showing GUS expression in ripening pollen grains as well as in germinating pollen. Moreover, most At TRXh is also expressed in pollen grains (Reichheld et al., 2002; Honys and Twell, 2004). Thus, both gene expression data and pollen competition data suggest that an NTS is functional in pollen. Proteomic analysis established a link between TRX and cytoplasmic male sterility by the identification of a mitochondrial aldehyde dehydrogenase as a potential target of TRX (Balmer et al., 2004). In maize, this protein was shown to be responsible for the restoration of cytoplasmic male sterility mediated by a polypeptide (URF13) that forms a mitochondrial inner membrane pore (Schnable and Wise, 1998; Liu et al., 2001). Morphological changes associated with cytoplasmic male sterility are generally associated with defects in anthers and tetrad development. Such defects are not observed in the ntra ntrb mutant. Still, mitochondrial functions often interfere with pollen development. Since NTRB is the major mitochondrial NTR, it is tempting to propose that the complete absence of mitochondrial NTR in ntra ntrb gametes is responsible for the defect in pollen fitness. A target protein specifically needed at that stage of development still needs to be identified. TRXs highly expressed in pollen could be good candidates.

A Glutathione-Dependent Pathway Reduces Type h Thioredoxins in the Absence of NTR and Allows Growth

The observation that a double mutation of the ntra and ntrb genes does not lead to severe developmental defects and possibly lethality was rather surprising, given that NTRs are supposed to be the unique reducing factors for cytosolic and mitochondrial thioredoxins. This is clearly not the case, because a substantial part of the cytosolic thioredoxin TRXh3 protein was found in a reduced state in the ntra ntrb mutant. The measurement of the redox state of TRXh3 under BSO treatment clearly indicates that glutathione is involved in this alternative reduction of thioredoxin, emphasizing crosstalk between the thioredoxin and glutathione systems. Nevertheless, a direct reduction of type h thioredoxins by glutathione is excluded by our biochemical data, which suggest that glutaredoxin may be responsible for the alternative reduction of thioredoxin in the ntra ntrb mutant. In vitro glutaredoxin-mediated reduction was shown previously for the poplar (Populus sp) cytosolic pop Trxh4, which is not reducible by NTRs (Gelhaye et al., 2003). This thioredoxin belongs to a subgroup distinct from TRXh3, suggesting that glutathione is able to reduce several types of type h thioredoxins. Nevertheless, our in vitro experiments on the reduction of TRXh3 by GRX1 and GRX4 demonstrate that these particular GRXs are less efficient than NTR in reducing TRXh. One possible explanation for an alternative reduction of TRX in planta is that other GRXs among the 30 to 40 GRXs and GRX homologs encoded by the Arabidopsis genome are more efficient for the reduction of TRX. Given the considerable similarity of plant GRs with animal thioredoxin reductases (Meyer et al., 2006), GR could account for the alternative reduction of TRX in the ntra ntrb mutant. Nevertheless, if the unique cytoplasmic GR reduces TRXs, this process would not be glutathione-dependent.

The involvement of both NTS and NGS pathways in maintaining the redox state of TRX has been described previously in budding yeast (Trotter and Grant, 2005). The redox state of the mitochondrial Trx3 was shown to be more affected in the double trr2 glr mutant than in the single trr2 mutant, in which the mitochondrial thioredoxin reductase TRR2 was inactivated, or in the glr mutant, in which both the cytosolic and mitochondrial isoforms of glutathione reductase GLR1 were inactivated. This suggests that a similar alternative thioredoxin reduction pathway occurs in yeast mitochondria. Therefore, it would be interesting to evaluate the redox state of the mitochondrial thioredoxin in the ntra ntrb mutant. Unfortunately, this cannot be easily evaluated by the AMS method that we use here, because the redox environment is not maintained during the mitochondrial purification steps.

The alternative reduction pathway masks most functions of TRXs, allowing almost normal activity of the TRXs in the ntra ntrb mutant. The simplest explanation for the phenotype of the ntra ntrb mutant compared with the wild type is that the growth retardation and abnormal seed morphology are due to the incomplete reduction of cytosolic TRXs by the alternative glutathione reduction. Nevertheless, we cannot completely exclude that NTRs may have targets other than the TRXs. Such proteins should present special characteristics: the redox center of the Arabidopsis NTRs is situated in a groove and is only accessible to proteins with a disulfide bridge on the surface of a small molecule or on an appendage of a large molecule (Dai et al., 1996). To date, such structures have been described only for the thioredoxin superfamily and, more recently, for the γ-interferon–inducible thiol reductases (Hastings et al., 2006), with six homologs in the Arabidopsis genome, one of them implicated in high O-acetyl-l-Ser accumulation (Ohkama-Ohtsu et al., 2004).

The Overlapping Role of NTR and Glutathione in Meristematic Activities

Although both NTRA and NTRB gene expression have been detected in meristematic tissues, the influence of both NTR inactivations has limited effect on meristematic activity. In fact, our data suggest that NTRs and glutathione function at different stages of meristem growth. Neither NTRs nor a high glutathione level appear to be necessary for root and shoot meristem formation during seed development, as shown by the presence of homozygous seeds in rml1. Even ntra ntrb rml1 homozygous seeds are able to sustain some growth on a glutathione medium, showing that their viability is not impaired. This notion was recently contradicted by the characterization of a knockout mutant in the GSH1 gene in which no seed formation takes place (Cairns et al., 2006). This mutant revealed that a small amount of GSH is necessary for embryo development. NTRs play a limited role in the root and apical meristem, leading only to a small reduction of growth in the ntra ntrb mutant. By contrast, the low synthesis of glutathione in rml1 completely blocks root meristem growth while the shoot meristem develops (Cheng et al., 1995; Vernoux et al., 2000). Shoot growth in rml1 is almost normal in the first days after germination but is clearly limited later, probably by the low nutrient supply caused by the absence of a root system. An important point is that shoot grown in rml1 is dependent on a functional NTR, as shown by the complete block of the apical meristem in ntra ntrb rml1. This strongly implicates thioredoxins as a key factor in shoot growth. They could be activated alternatively by NTRs or by a high level of glutathione.

By contrast, root growth cannot be sustained at a low level of glutathione. This does not exclude thioredoxin function in the root meristem but suggests that low glutathione limits root growth in a thioredoxin-independent manner. The partial recovery of meristematic activity in the ntra ntrb rml1 mutant growing on GSH medium strongly suggests that the developmental defect results from modulation of upstream signal transduction pathways rather than from a deleterious accumulation of activated oxygen species in meristematic cells. Meristem activity and cellular proliferation have been shown to be controlled by regulatory genes that are expressed in specific subdomains of the meristem. Among these genes, homeodomain proteins like SHOOTMERISTEMLESS, WUSCHEL, CLAVATA1 and CLAVATA3, and STIMPY are required for shoot apical meristem maintenance. Other transcription factors like SCARECROW regulate the root apical meristem. Whether such regulatory proteins could be under redox regulation has to be considered. A number of transcription factors have been shown to be redox-dependent, but no data are yet available about redox-dependent factors implicated in meristematic identity.

Recently, Jiang et al. (2006) reported a detailed analysis of the redox potentials in the Arabidopsis root using reporter plants expressing a redox-sensing green fluorescent protein. The root cap and meristem have a more reduced redox status than the elongation zone. These data are in agreement with our data showing expression of NTR in the root meristem and root cap and with the role of the NTS and glutathione pathways in root meristematic activity. Redox-sensing green fluorescent protein constructs would be a powerful tool to study the redox status in our mutant backgrounds.

The ntra ntrb Mutant Is Not Hypersensitive to Oxidants

Thioredoxins are generally considered to be important antioxidants. This function was clearly demonstrated by genetic means for some chloroplast thioredoxins (Vieira Dos Santos and Rey, 2006). An antioxidant function of the NTS is suggested even in the cytosol and mitochondria by the presence of the misnamed plant glutathione peroxidases (GPXs), which are in fact thioredoxin-dependent peroxidases (Iqbal et al., 2006; Navrot et al., 2006), and of peroxiredoxins (PRXs), some reducible by thioredoxins (Brehelin et al., 2003) and others by glutaredoxins (Dietz, 2003). The simplest interpretation is that the alternative thioredoxin reduction pathway is sufficient to allow optimal function of the GPXs and PRXs in the ntra ntrb mutant. More surprising was the reversibility in growth observed with the BSO-treated ntra ntrb mutant when transferred to BSO-free medium. The treated seedlings do not suffer from a toxic accumulation of activated oxygen species. Similarly, triple mutant ntra ntrb rml1 seedlings grow in the presence of exogenously applied glutathione, showing their viability. Thus, the cytosolic and mitochondrial GPXs and PRXs may only be involved in more severe biotic or abiotic situations that generate massive amounts of activated oxygen species. In addition, it is now well established that some GPXs are oxidative stress sensors rather than direct antioxidants (Miao et al., 2006). Moreover, the high anthocyanin content observed in the ntra ntrb mutant could be an adaptation to an alternative antioxidant defense.

METHODS

Plant Materials, Growth Conditions, and Treatments

Seedlings and plants from Arabidopsis thaliana ecotype Col-0 (wild-type, rml1, and SALK mutant lines) were used for the experiments. T-DNA mutant lines were provided by the SALK laboratory (Alonso et al., 2003) (http://www.Arabidopsis.org). For in vitro seedlings, seeds were surface-sterilized and plated on half-strength MS medium including Gamborg B5 vitamins (M0231; Duchefa), 1% (w/v) sucrose, and 0.8% (w/v) plant agar. Supplementation of the growth medium with BSO (Sigma-Aldrich), GSH (Sigma-Aldrich), GSSG (Sigma-Aldrich), lycorine (Sigma-Aldrich), H₂O₂ (Sigma-Aldrich), and menadione (Sigma-Aldrich) at various concentrations was performed as follows. Appropriate amounts of the filter-sterilized stock solution containing these substances were added to sterile Petri dishes, and molten half-strength MS medium that had been cooled to 50°C was added while swilling the plates. For root growth measurements, seeds were germinated on vertical plates, and the position of the root tip was marked on the underside of the plate. Every day after the transfer, root growth was measured on 20 to 30 roots per plate using a binocular microscope (Leika MZ12) with the aid of an eyepiece graticule. For plant growth in soil, seeds were sown in pots containing a mixture of soil and vermiculite (3:1, v/v) and irrigated with water. Both plants and seedlings were grown at 22°C and 70% hygrometry under a 16-h light (4,500 to 10,000 lux)/8-h dark regime.

Microscopic Observations and Tissue Cross Sections

For phenotypic characterization and cell density determination, tissues were cleared overnight with Hoyer's solution (2.5 g of arabic gum, 100 g of chloral hydrate, 5 mL of glycerol, and 30 mL of distilled water), mounted on slides, and observed with a binocular microscope (Zeiss Axioskop2). Pictures were taken with a numeric camera (Leica DC300FX) coupled to an imaging software (Leica FW4000). Cross sections of leaves were prepared using a microtome (Leica RM2255) from tissues embedded in hydroxyethyl methacrylate (Technovit 7100; Heraus-Kulter) and were counterstained in purple with periodic acid Schiff reagents.

Pollen Competition and Mutant Crosses

Pollen competition experiments were performed by emasculation of flower buds of the ntra ntrb mutant followed by pollinization with ripening pollen grains of mature flowers of plants of distinct genotypes. Siliques were allowed to grow until seed maturation was complete. In order to discriminate between homozygote ntra ntrb and heterozygote ntra NTRB, seeds were selected on MS/2 medium supplemented by 0.5 mM BSO. The same method was used for crossing ntra, ntrb, and rml1 mutants.

Gene Expression Analysis by RT-PCR

Total RNA was extracted from frozen plant organs using TRIzol reagent (Gibco BRL) according to the manufacturer's protocol. For RT-PCR, 5 μg of DNase I–treated total RNA was used for first-strand synthesis of cDNA using oligo(dT) primer reverse transcription with the Moloney murine leukemia virus reverse transcriptase as described by the manufacturer's protocol (First-Strand RT-PCR kit, ProSTAR; Stratagene). PCR (25 cycles) was performed as described by Laloi et al. (2004). The following primers were used for RT-PCR experiments: NTRA forward primer, 5′-GCAAAATGTGTTGGATCTCAATGAG-3′, and reverse primer, 5′-CATGGATCCTTCTCCTACAGCTTC-3′; NTRB forward primer, 5′-CGAAAGCTTTGCACGGCTTGGTGGTG-3′, and reverse primer, 5′-GATCAATCAACAATAACTCAATGACCT-3′; Act2 forward primer, 5′-GTTAGCAACTGGGATGATATGG-3′, and reverse primer, 5′-AGCACCAATCGTGATGACTTGCCC-3′.

Cloning of Complementation and GUS Expression Constructs: Expression Analysis Using the GUS Reporter Gene

For complementation experiments with the NTRB gene, a genomic fragment of 3270 bp (1497 bp upstream from ATG2 to 381 bp downstream from the stop codon) was amplified by PCR using primers introducing unique XhoI and XbaI sites (5′-CGCTCGAGGTAGCAGAAGAAGGTGAAGAAGAAG-3′ and 5′-CCTCTAGAAGCTCTGGAAATGACTCGGCTTC-3′, respectively). After sequencing, the fragment was cloned into the pCAMBIA-1300-HYG binary vector at the compatible SalI and XbaI sites.

For differential complementation with the cytosolic and mitochondrial forms of NTRB, specific NTRB cDNAs were amplified by PCR on total cDNAs prepared from Arabidopsis Col-0 plantlets using GATEWAY-compatible primers (Invitrogen). In order to express the cytosolic isoform of the NTRB protein, the NTRB cDNA was cloned at the more downstream ATG (ATG2) using the following primers (ATG2, 5′-GGAGATAGAACCATGAACTGTGTGAGTCGTTTAAAGTG-3′, and stop, 5′-GGAGATAGAACCATGAATGGTCTCGAAACTCACAACACAAGG-3′). To express the mitochondrial isoform, the cDNA was cloned at the upstream ATG (ATG1) using the following primers (ATG1, 5′-GGAGATAGAACCATGAATGGTCTCGAAACTCACAACACAAGG-3′, and the same stop primer as above). These transformants were called NTRBc and NTRBm, respectively. Both constructs were cloned into the pDON207 vector and sequenced. To avoid initiation of the cytosolic isoform in the NTRBm construct, ATG2 was mutated to ATC using the QuickChange site-directed mutagenesis kit (Stratagene) with the following upstream and downstream primers (5′-CTTCATCCGCCGTCATCAATGGTCTCGAAAGTG-3′ and 5′-CACTTTCGAGACCATTGATGACGGCGGATGTTG-3′, respectively). Both constructs were cloned by recombination in the pH7WG2D binary vector (Invitrogen).

For GUS expression analyses, fragments of 1528 and 1492 bp of the regions upstream from the ATG codons of NTRA and NTRB, respectively, were isolated by direct PCR on genomic DNA using primers to introduce unique XhoI and NcoI sites. The DNA fragments were then digested by the corresponding enzymes and cloned into SalI- and NcoI-compatible sites of pCAMBIA1304-HYG binary vector.

The resulting plasmids were introduced into Agrobacterium tumefaciens C18CIRif^R. Arabidopsis plants (Col-0 or ntra ntrb mutant) were transformed with agrobacteria by the floral dip method (Clough and Bent, 1998). T1, T2, and T3 seedlings were selected in vitro on MS/2 medium supplemented with 30 μg/mL hygromycin. For the GUS assay, plants were either cultivated in vitro under the same conditions or grown in soil mixed with vermiculite in a greenhouse under continuous light at 22°C. GUS histochemical staining was performed according to Lagarde et al. (1996).

Preparation of Mitochondrial and Cytosolic Extracts from Arabidopsis Plants

Stems with flowers and siliques from 8-week-old Arabidopsis plants were used for organelle preparation as described by Reichheld et al. (2005). Briefly, the aerial tissue was disrupted in extraction buffer using a Waring blender (25 mL/5 g material). Four centrifugations were performed to eliminate particles with low sedimentation coefficients, followed by a final pelleting of a mitochondria-enriched fraction (at 17,000g). The supernatant of the 17,000g centrifugation was further centrifuged at 100,000g, and the soluble fraction was kept as a cytosolic fraction. The mitochondrial pellets were further purified on 18 to 29 to 45% Percoll step gradients. The purified mitochondria were collected at the 29 to 45% interphase.

Plant Protein Extraction, Protein Gel Blot Analysis, and Immunodetection

Unless stated otherwise, Arabidopsis protein extracts were prepared by grinding tissues in liquid nitrogen and resuspension in extraction buffer (25 mM Tris-HCl, pH 7.6, 75 mM NaCl, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40). After centrifugation (15 min, 13,000 rpm, 4°C), protein concentrations from the supernatant were determined using the Protein Assay kit (Bio-Rad). Proteins were separated by SDS-PAGE and transferred to Immobilon-P membranes (Amersham Pharmacia). Rabbit polyclonal antibodies against NTRB were diluted 1:10,000 for protein gel blotting. Antibodies directed against wheat subunit 9 of NADH dehydrogenase (Lamattina et al., 1993) were used as a control for the mitochondrial protein fraction. Goat anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham Pharmacia) were used as secondary antibodies and revealed with enhanced chemiluminescence reagents (Amersham Pharmacia).

Enzymatic Activities

Thioredoxin activity was determined using the insulin-disulfide reduction assay as described (Laloi et al., 2001). The reaction was initiated by the addition of 0.13 mM bovine insulin (Sigma-Aldrich) to 0.5 mL of 0.1 M potassium phosphate buffer, pH 7.0, containing 2 mM EDTA, 0.5 mM NADPH (Sigma-Aldrich), different concentrations of At TRXh3, and the GRX system composed of 0.5 units of GR (Roche Diagnostics), 0.5 mM GSH (Sigma-Aldrich), and 5 μM recombinant At GRX1 or At GRX4 (C. Riondet and J.-P. Desouris, unpublished data). The TRX system, composed of 5 μM TRXh3 and 5 μM NTRA, was used as a control. The consumption of NADPH was followed spectrophotometrically at 340 nm at 22°C.

Glutathione and Anthocyanin Determination

Glutathione was determined using monobromobimane (Calbiochem) as reported in the procedure of Fahey and Newton (1987). Liquid nitrogen plant powder extracts were mixed in the presence of 50 mM HCl and then centrifuged at full speed to obtain a clear supernatant. A volume of the supernatant was then mixed in the presence of 0.05 mL of 1 M HEPES, pH 8.2, with 0.002 mL of 100 mM DTT and 0.002 mL of 200 mM Na₂-EDTA.The mixture was incubated for 10 min prior to the addition of 0.008 mL of 100 mM monobromobimane in acetonitrile. After 10 min of incubation, the reaction was stopped with 0.040 mL of methane sulfonic acid. Derivatives were separated by reverse chromatography with HPLC (Waters) using an Uptisphere column (5μ HDO C18, 250 × 4.6 cm; Interchim). Elution was performed with buffer A (0.25% [v/v] acetic acid, 5% [v/v] acetonitrile, and 57 mM sodium perchlorate, pH 3.4) and buffer B (0.25% [v/v] acetic acid and 80% [v/v] acetonitrile). Separation was performed for 25 min at 1 mL/min using the following gradient: 0 min, A + 5% B; 5 min, A + 12% B; 18 min, A + 12% B; 18 min, 100% B; 20 min, 100% B; and then reequilibration for 15 min with A + 5% B before the next injection. Derivatives were detected at 480 nm after excitation at 388 nm using an SFM25 fluorimeter. In this condition, GSH was eluted at a single peak at 17.8 min.

The anthocyanin content was evaluated as reported before (Feinbaum and Ausubel, 1988). Tissues were blended in liquid nitrogen and mixed in the presence of 1 volume of ethanol containing 1% HCl. After mixing, 1 volume of methanol:water (1:3) was added, followed by 1 volume of chloroform. After centrifugation at full speed to pellet membranes and precipitated proteins, the clear supernatant was mixed with 1 volume of methanol:water (1:3). Absorbance at 530 nm was measured to evaluate the amount of anthocyanins in all samples.

Redox State of TRXh3 by AMS Treatment

The redox state of TRXh3 was determined as described (Ritz and Beckwith, 2002) with some modifications. Arabidopsis plantlets were ground in liquid nitrogen, and the fine powder was dissolved in 5% trichloroacetic acid. After filtration on a 60-μm nylon net filter (NY60; Millipore) to remove tissue fragments, proteins were precipitated for 1 h on ice. Protein extracts were collected after centrifugation for 15 min, 13,000 rpm at 4°C. Pellets were washed with ice-cold acetone, dried, and dissolved in 200 mM Tris-HCl, pH 7.5, and 2% SDS. Cys residues were alkylated by 15 mM AMS for 1 h at room temperature in the dark. Proteins were separated by 15% SDS-PAGE under nonreductive conditions as described previously (Serrato and Cejudo, 2003). At TRXh3 protein was visualized by Western blot with anti-TRXh3 antibodies.

Database Searches and Sequence Analysis

DNA sequences were analyzed using a locally installed BLAST server (Altschul et al., 1997) against public and local nucleotide and protein databases. The physical locations of genes in the Arabidopsis genome were determined using the maps available at The Arabidopsis Information Resource (http://www.Arabidopsis.org/). Multiple sequence analysis was performed using the ClustalW software (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html).

Accession Numbers

Sequence data for NTRA, NTRB, TRXh3, GRX1, GRX4, and ACT2 can be found in the GenBank/EMBL data libraries under accession numbers NM 127297 (At2g17420), Z23109.1 (At4g35460), NM 123664 (At5g42980), NM 123404.2 (At5g40370), NM 125697.1 (At5g63030), and NM 112764.2 (At3g18780), respectively.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Delayed Bolting and Slower Inflorescence Stem Growth in the ntra ntrb Mutant.
Supplemental Table 1. Glutathione Level and the Redox State of Glutathione Are Not Modified in the ntra ntrb Mutant.

Supplementary Material

[Supplemental Data]

Click here to view.

Acknowledgments

We thank the ABRC for T-DNA insertional mutants, Y. Moné and Y. Yao for help with complementation constructs and analyses, D. Roby for pathogen-challenging experiments, and R. Cooke for critical reading of the manuscript. This work was supported by funding from GENOPLANTE 2.

Notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Jean-Philippe Reichheld (jpr/at/univ-perp.fr).

^[W]Online version contains Web-only data.

www.plantcell.org/cgi/doi/10.1105/tpc.107.050849

References

Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657. [PubMed].
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402. [PubMed].
Arabidopsis Genome Initiative (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408: 796–815. [PubMed].
Arner, E.S., and Holmgren, A. (2000). Physiological functions of thioredoxin and thioredoxin reductase. Eur. J. Biochem. 267: 6102–6109. [PubMed].
Balmer, Y., Vensel, W.H., Tanaka, C.K., Hurkman, W.J., Gelhaye, E., Rouhier, N., Jacquot, J.-P., Manieri, W., Schurmann, P., Droux, M., and Buchanan, B.B. (2004). Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria. Proc. Natl. Acad. Sci. USA 101: 2642–2647. [PubMed].
Brehelin, C.M., Meyer, E.H., de Souris, J.-P., Bonnard, G., and Meyer, Y. (2003). Resemblance and dissemblance of Arabidopsis type II peroxiredoxins: Similar sequences for divergent gene expression, protein localization, and activity. Plant Physiol. 132: 2045–2057. [PubMed].
Cairns, N.G., Pasternak, M., Wachter, A., Cobbett, C.S., and Meyer, A.J. (2006). Maturation of Arabidopsis seeds is dependent on glutathione biosynthesis within the embryo. Plant Physiol. 141: 446–455. [PubMed].
Carmel-Harel, O., and Storz, G. (2000). Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu. Rev. Microbiol. 54: 439–461. [PubMed].
Cheng, J.-C., Seeley, K.A., and Sung, Z.R. (1995). RML1 and RML2, Arabidopsis genes required for cell proliferation at the root tip. Plant Physiol. 107: 365–376. [PubMed].
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743. [PubMed].
Dai, S., Saarinen, M., Ramaswamy, S., Meyer, Y., Jacquot, J.P., and Eklund, H. (1996). Crystal structure of Arabidopsis thaliana NADPH dependent thioredoxin reductase at 2.5 A resolution. J. Mol. Biol. 264: 1044–1057. [PubMed].
Dai, S., Schwendtmayer, C., Schurmann, P., Ramaswamy, S., and Eklund, H. (2000). Redox signaling in chloroplasts: Cleavage of disulfides by an iron-sulfur cluster. Science 287: 655–658. [PubMed].
Dietz, K.J. (2003). Plant peroxiredoxins. Annu. Rev. Plant Biol. 54: 93–107. [PubMed].
Draculic, T., Dawes, I.W., and Grant, C.M. (2000). A single glutaredoxin or thioredoxin gene is essential for viability in the yeast Saccharomyces cerevisiae. Mol. Microbiol. 36: 1167–1174. [PubMed].
Fahey, R.C., and Newton, G.L. (1987). Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Methods Enzymol. 143: 85–96. [PubMed].
Feinbaum, R.L., and Ausubel, F.M. (1988). Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol. Cell. Biol. 8: 1985–1992. [PubMed].
Gelhaye, E., Rouhier, N., and Jacquot, J.P. (2003). Evidence for a subgroup of thioredoxin h that requires GSH/Grx for its reduction. FEBS Lett. 555: 443–448. [PubMed].
Hastings, K.T., Lackman, R.L., and Cresswell, P. (2006). Functional requirements for the lysosomal thiol reductase GILT in MHC class II-restricted antigen processing. J. Immunol. 177: 8569–8577. [PubMed].
Holmgren, A. (1985). Thioredoxin. Annu. Rev. Biochem. 54: 237–271. [PubMed].
Honys, D., and Twell, D. (2004). Transcriptome analysis of haploid male gametophyte development in Arabidopsis. Genome Biol. 5: R85. [PubMed].
Iqbal, A., Yabuta, Y., Takeda, T., Nakano, Y., and Shigeoka, S. (2006). Hydroperoxide reduction by thioredoxin-specific glutathione peroxidase isoenzymes of Arabidopsis thaliana. FEBS J. 273: 5589–5597. [PubMed].
Jakupoglu, C., Przemeck, G.K.H., Schneider, M., Moreno, S.G., Mayr, N., Hatzopoulos, A.K., de Angelis, M.H., Wurst, W., Bornkamm, G.W., Brielmeier, M., and Conrad, M. (2005). Cytoplasmic thioredoxin reductase is essential for embryogenesis but dispensable for cardiac development. Mol. Cell. Biol. 25: 1980–1988. [PubMed].
Jiang, K., Schwarzer, C., Lally, E., Zhang, S., Ruzin, S., Machen, T., Remington, S.J., and Feldman, L. (2006). Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis. Plant Physiol. 141: 397–403. [PubMed].
Karimpour, S., et al. (2002). Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation. Oncogene 21: 6317–6327. [PubMed].
Keryer, E., Collin, V., Lavergne, D., Lemaire, S., and Issakidis-Bourguet, E. (2004). Characterization of Arabidopsis mutants for the variable subunit of ferredoxin:thioredoxin reductase. Photosynth. Res. 79: 265–274. [PubMed].
Koc, A., Mathews, C.K., Wheeler, L.J., Gross, M.K., and Merrill, G.F. (2006). Thioredoxin is required for deoxyribonucleotide pool maintenance during S phase. J. Biol. Chem. 281: 15058–15063. [PubMed].
Lagarde, D., Basset, M., Lepetit, M., Conejero, G., Gaymard, F., Astruc, S., and Grignon, C. (1996). Tissue-specific expression of Arabidopsis AKT1 gene is consistent with a role in K⁺ nutrition. Plant J. 9: 195–203. [PubMed].
Laloi, C., Mestres-Ortega, D., Marco, Y., Meyer, Y., and Reichheld, J.P. (2004). The Arabidopsis cytosolic thioredoxin h5 gene induction by oxidative stress and its W-box-mediated response to pathogen elicitor. Plant Physiol. 134: 1006–1016. [PubMed].
Laloi, C., Rayapuram, N., Chartier, Y., Grienenberger, J.M., Bonnard, G., and Meyer, Y. (2001). Identification and characterization of a mitochondrial thioredoxin system in plants. Proc. Natl. Acad. Sci. USA 98: 14144–14149. [PubMed].
Lamattina, L., Gonzalez, D., Gualberto, J., and Grienenberger, J.M. (1993). Higher plant mitochondria encode an homologue of the nuclear-encoded 30-kDa subunit of bovine mitochondrial complex I. Eur. J. Biochem. 217: 831–838. [PubMed].
Liu, F., Cui, X., Horner, H.T., Weiner, H., and Schnable, P.S. (2001). Mitochondrial aldehyde dehydrogenase activity is required for male fertility in maize. Plant Cell 13: 1063–1078. [PubMed].
Matsui, M., Oshima, M., Oshima, H., Takaku, K., Maruyama, T., Yodoi, J., and Taketo, M.M. (1996). Early embryonic lethality caused by targeted disruption of the mouse thioredoxin gene. Dev. Biol. 178: 179–185. [PubMed].
Maughan, S., and Foyer, C.H. (2006). Engineering and genetic approaches to modulating the glutathione network in plants. Physiol. Plant. 126: 382–397.
Meyer, Y., Riondet, C., Constans, L., Abdelgawwad, M.R., Reichheld, J.P., and Vignols, F. (2006). Evolution of redoxin genes in the green lineage. Photosynth. Res. 89: 1–14. [PubMed].
Miao, Y., Lv, D., Wang, P., Wang, X.C., Chen, J., Miao, C., and Song, C.P. (2006). An Arabidopsis glutathione peroxidase functions as both a redox transducer and a scavenger in abscisic acid and drought stress responses. Plant Cell 18: 2749–2766. [PubMed].
Navrot, N., Collin, V., Gualberto, J., Gelhaye, E., Hirasawa, M., Rey, P., Knaff, D.B., Issakidis, E., Jacquot, J.-P., and Rouhier, N. (2006). Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses. Plant Physiol. 142: 1364–1379. [PubMed].
Nishinaka, Y., Nakamura, H., and Yodoi, J. (2002). Thioredoxin cytokine action. Methods Enzymol. 347: 332–338. [PubMed].
Nordberg, J., and Arner, E.S. (2001). Reactive oxygen species, antioxidants, and the mammalian thioredoxin system. Free Radic. Biol. Med. 31: 1287–1312. [PubMed].
Ohkama-Ohtsu, N., Kasajima, I., Fujiwara, T., and Naito, S. (2004). Isolation and characterization of an Arabidopsis mutant that overaccumulates O-acetyl-L-Ser. Plant Physiol. 136: 3209–3222. [PubMed].
Perez-Ruiz, J.M., Spinola, M.C., Kirchsteiger, K., Moreno, J., Sahrawy, M., and Cejudo, F.J. (2006). Rice NTRC is a high-efficiency redox system for chloroplast protection against oxidative damage. Plant Cell 18: 2356–2368. [PubMed].
Potters, G., Horemans, N., Bellone, S., Caubergs, R.J., Trost, P., Guisez, Y., and Asard, H. (2004). Dehydroascorbate influences the plant cell cycle through a glutathione-independent reduction mechanism. Plant Physiol. 134: 1479–1487. [PubMed].
Qin, J., Clore, G.M., Kennedy, W.M., Huth, J.R., and Gronenborn, A.M. (1995). Solution structure of human thioredoxin in a mixed disulfide intermediate complex with its target peptide from the transcription factor NF kappa B. Structure 3: 289–297. [PubMed].
Reichheld, J.P., Mestres-Ortega, D., Laloi, C., and Meyer, Y. (2002). The multigenic family of thioredoxin h in Arabidopsis thaliana: Specific expression and stress response. Plant Physiol. Biochem. 40: 685–690.
Reichheld, J.-P., Meyer, E., Khafif, M., Bonnard, G., and Meyer, Y. (2005). AtNTRB is the major mitochondrial thioredoxin reductase in Arabidopsis thaliana. FEBS Lett. 579: 337–342. [PubMed].
Ritz, D., and Beckwith, J. (2002). Redox state of cytosolic thioredoxin. Methods Enzymol. 347: 360–370. [PubMed].
Rivas, S., Rougon-Cardoso, A., Smoker, M., Schauser, L., Yoshioka, H., and Jones, J.D. (2004). CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defence. EMBO J. 23: 2156–2165. [PubMed].
Rouhier, N., Gelhaye, E., and Jacquot, J.-P. (2004). Plant glutaredoxins: Still mysterious reducing systems. Cell. Mol. Life Sci. 61: 1266–1277. [PubMed].
Rouhier, N., Gelhaye, E., Sautière, P.E., Brun, A., Laurent, P., Tagu, D., Gerard, J., de Fay, E., Meyer, Y., and Jacquot, J.-P. (2001). Isolation and characterization of a new peroxiredoxin from poplar sieve tubes that uses either glutaredoxin or thioredoxin as a proton donor. Plant Physiol. 127: 1299–1309. [PubMed].
Ruelland, E., and Miginiac-Maslow, M. (1999). Regulation of chloroplast enzyme activities by thioredoxin: Activation or relief from inhibition. Trends Plant Sci. 4: 136–141. [PubMed].
Russo, T., Zambrano, N., Esposito, F., Ammendola, R., Cimino, F., Fiscella, M., Jackman, J., O'Connor, P.M., Anderson, C.W., and Appella, E. (1995). A p53-independent pathway for activation of WAF1/CIP1 expression following oxidative stress. J. Biol. Chem. 270: 29386–29391. [PubMed].
Sanchez-Fernandez, R., Fricker, M., Corben, L.B., White, N.S., Sheard, N., Leaver, C.J., Van Montagu, M., Inze, D., and May, M.J. (1997). Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control. Proc. Natl. Acad. Sci. USA 94: 2745–2750. [PubMed].
Schnable, P.S., and Wise, R.P. (1998). The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci. 3: 175–180.
Schürmann, P., and Jacquot, J.P. (2000). Plant thioredoxin systems revisited. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 371–400. [PubMed].
Serrato, A., Perez-Ruiz, J., Spinola, M., and Cejudo, F. (2004). A novel NADPH thioredoxin reductase, localized in the chloroplast, which deficiency causes hypersensitivity to abiotic stress in Arabidopsis thaliana. J. Biol. Chem. 279: 43821–43827. [PubMed].
Serrato, A.J., and Cejudo, F.J. (2003). Type-h thioredoxins accumulate in the nucleus of developing wheat seed tissues suffering oxidative stress. Planta 217: 392–399. [PubMed].
Sohn, J., and Rudolph, J. (2003). Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction. Biochemistry 42: 10060–10070. [PubMed].
Trotter, E.W., and Grant, C.M. (2003). Non-reciprocal regulation of the redox state of the glutathione-glutaredoxin and thioredoxin systems. EMBO Rep. 4: 184–188. [PubMed].
Trotter, E.W., and Grant, C.M. (2005). Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae. Eukaryot. Cell 4: 392–400. [PubMed].
Verdoucq, L., Vignols, F., Jacquot, J.P., Chartier, Y., and Meyer, Y. (1999). In vivo characterization of a thioredoxin h target protein defines a new peroxiredoxin family. J. Biol. Chem. 274: 19714–19722. [PubMed].
Vernoux, T., Wilson, R.C., Seeley, K.A., Reichheld, J.P., Muroy, S., Brown, S., Maughan, S.C., Cobbett, C.S., Van Montagu, M., Inze, D., May, M.J., and Sung, Z.R. (2000). The ROOT MERISTEMLESS1/CADMIUM SENSITIVE2 gene defines a glutathione-dependent pathway involved in initiation and maintenance of cell division during postembryonic root development. Plant Cell 12: 97–110. [PubMed].
Vieira Dos Santos, C., and Rey, P. (2006). Plant thioredoxins are key actors in the oxidative stress response. Trends Plant Sci. 11: 329–334. [PubMed].