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First published online July 9, 2008; 10.1104/pp.108.123778 Plant Physiology 148:424-435 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
A Novel Type of Thioredoxin Dedicated to Symbiosis in Legumes1,[W],[OA]Physiologie Moléculaire des Semences, UMR 1191 Université d'Angers-Institut National d'Horticulture-INRA, IFR 149 QUASAV, ARES, 49045 Angers cedex 01, France (F.A., M.R., F.M.); Interactions Biotiques et Santé Végétale, UMR INRA 1301/CNRS 6243 Université de Nice-Sophia Antipolis, Centre de Recherche INRA de Sophia Antipolis, 06903 Sophia Antipolis cedex, France (P.F.); Institut de Biologie Moléculaire des Plantes, laboratoire propre du CNRS (UPR 2357) conventionné avec l'Université Louis Pasteur (Strasbourg 1), 67084 Strasbourg cedex, France (C.K., C.R.); Génome et Développement des Plantes, UMR 5096 Université de Perpignan-CNRS, 66860 Perpignan, France (Y.M.); Interaction Arbres/Micro-organismes, UMR 1136 Université Nancy I-INRA, Faculté des Sciences, 54506 Vandoeuvre, France (E.G.); and Department of Chemistry and Biochemistry and Center for Biotechnology and Genomics, Texas Tech University, Lubbock, Texas 79409–1061 (M.H., D.B.K.)
Thioredoxins (Trxs) constitute a family of small proteins in plants. This family has been extensively characterized in Arabidopsis (Arabidopsis thaliana), which contains six different Trx types: f, m, x, and y in chloroplasts, o in mitochondria, and h mainly in cytosol. A detailed study of this family in the model legume Medicago truncatula, realized here, has established the existence of two isoforms that do not belong to any of the types previously described. As no possible orthologs were further found in either rice (Oryza sativa) or poplar (Populus spp.), these novel isoforms may be specific for legumes. Nevertheless, on the basis of protein sequence and gene structure, they are both related to Trxs m and probably have evolved from Trxs m after the divergence of the higher plant families. They have redox potential values similar to those of the classical Trxs, and one of them can act as a substrate for the M. truncatula NADP-Trx reductase A. However, they differ from classical Trxs in that they possess an atypical putative catalytic site and lack disulfide reductase activity with insulin. Another important feature is the presence in both proteins of an N-terminal extension containing a putative signal peptide that targets them to the endoplasmic reticulum, as demonstrated by their transient expression in fusion with the green fluorescent protein in M. truncatula or Nicotiana benthamiana leaves. According to their pattern of expression, these novel isoforms function specifically in symbiotic interactions in legumes. They were therefore given the name of Trxs s, s for symbiosis.
Thioredoxins (Trxs) are small and ubiquitous proteins with two close and active redox-active Cys residues in a conserved WCG/PPC motif. In their dithiol form, they are powerful disulfide reductases (Holmgren, 1985  ) that play a posttranslational regulatory role in proteins involved in an ever-increasing number of cellular processes (Buchanan and Balmer, 2005). In plants, Trxs constitute a small protein family. Twenty-two genes have been detected in the fully sequenced Arabidopsis (Arabidopsis thaliana) genome, including two encoding monocysteinic isoforms (WCXXS) first described in this species (Meyer et al., 2002, 2005) but also present in poplar (Populus spp.; Gelhaye et al., 2003, 2004a). In contrast, the genomes of Escherichia coli, Saccharomyces cerevisiae, and Homo sapiens contain only two to three genes, all of which encode bicysteinic isoforms.
The plant Trx family is divided into six different types according to the primary structure and localization of its members: the f, m, x, and y types are in the chloroplasts (Buchanan, 1991
Despite the existence of numerous isoforms in each type and some redundancy in their functions, some plant Trx isoforms were found to be preferentially involved in interactions of plants with both pathogenic and symbiotic microorganisms. Indeed, a Trx x isoform in tomato (Solanum lycopersicum) plants was shown to render the interaction of tomato with Cladosporium fulvum compatible by decreasing the defense response through its interaction with the cf-9 protein (Rivas et al., 2004 The Trx family has been most intensively studied in the model species Arabidopsis. However, whether the phylogeny and function of Trxs in other plants are similar to those reported for Arabidopsis remains to be established. Here, we have taken advantage of the growing body of knowledge concerning the genes and genome of the model Medicago truncatula, a galegoid legume, to characterize the Trx family in leguminous plants. In contrast to Arabidopsis, legumes are characterized by symbiotic relationships with both nitrogen-fixing bacteria and mycorrhizal fungi. For that purpose, Trx sequences were searched in EST and genomic databases and compared to those of Arabidopsis. While M. truncatula contains members of each of the Trx types known to be present in Arabidopsis, it also contains two isoforms of a novel type. This novel type, which to our knowledge has not been described previously, contains both an atypical catalytic site and a signal peptide. Because these novel isoforms, described below, seem to be dedicated to symbiotic interactions in legumes, they were given the name of Trxs s, s for symbiosis.
All the Types of Trxs Described for Arabidopsis Are Present in M. truncatula While the sequencing of M. truncatula genome is currently in progress (the release of the complete genome will only be effective by the end of the year 2008), large public EST databases are already available (MtGI, http//www.tigr.org/tdb/mtgi; MtDB, http://www.medicago.org). It is noteworthy that, at its last release (January 19, 2005), MtGI notably contained 226,923 ESTs in 36,878 unique sequences arising from various tissues of plants grown under either optimal or suboptimal conditions and in both the presence and the absence of symbiotic interactions. From these libraries, we were able to identify by homology searches about 80 sequences, tentative consensus (TC) or singletons, related to Trxs, among which 20 sequences of putative Trxs were identified (Table I ). Eighteen encode complete open reading frames, while two (a Trx o and a Trx x) are only partially complete. Two additional Trx sequences were encountered in genomic data (Table I). All the Trx sequences were aligned with ClustalW and then compared to those of Arabidopsis in a phylogenetic tree (Fig. 1 ). All the types of Trxs previously described for Arabidopsis are present in M. truncatula. While some types are represented in M. truncatula by only a single member, others contain multiple isoforms: one Trx f, four Trxs m, one Trx x, one Trx y, one Trx o, and 12 Trxs h. However, M. truncatula also seems to have an additional type of Trx with two isoforms that forms a distinct cluster in the tree (see below).
A Novel Type of Trx Exists in M. truncatula As mentioned above, two sequences that do not seem to belong to the types of Trxs described so far were found in M. truncatula EST data banks. The deduced sequences of these two proteins share 53% identity and form a distinct cluster in the phylogenetic tree (Fig. 1) that is close to the cluster of isoforms of the m type, which explains their annotation as m type in the databases. As the corresponding genomic sequences are not yet known, further analysis required the cloning of the genes from M. truncatula. As in the case of Arabidopsis Trxs m, the gene sequences have only one intron that separates the regions encoding a putative transit peptide and the rest of the protein (Supplemental Fig. S1). We also cloned their coding regions (DQ121444, DQ121445). The deduced protein sequences are shown in Supplemental Figure S1 and Figure 2 . Comparison with the sequence of Trx h2 (DQ121443), a Trx of h type also cloned in our study, reveals that both proteins have an N-terminal extension that may correspond to a transit peptide (Fig. 2). This extension, which is highly similar for the two isoforms (20 out of 27 first amino acids are identical), is nevertheless shorter than the chloroplast target peptides of Trxs m (Fig. 2) and does not contain the charge characteristics required for a chloroplast import. In contrast, according to the predictions of different computer programs (Predotar, SignalP, TargetP, Psort), it could correspond to a signal peptide that targets the proteins to the endoplasmic reticulum (ER). The potential cleavage site of the signal peptide in each isoform sequence is indicated in Supplemental Figure S1. To our knowledge, this is the first example of Trxs harboring a transit peptide of this nature have been encountered. In addition, the two isoforms contain an atypical putative catalytic site, LCSPC or WCGQNC, that differs from that of all previously described Trx types.
Because of the similarity of the two isoforms to Trxs m (at both the level of gene structure and protein sequence), these isoforms appeared to be bona fide members of the Trx family. Nevertheless, because of the presence in their sequences of both an atypical active site and a predicted signal peptide, they clearly define a novel Trx type. They were given the name of Trxs s for symbiosis, because the corresponding ESTs were present only in symbiotic root libraries of M. truncatula, interacting with Sinorhizobium meliloti (s1, eight ESTs; s2, eight ESTs) or Glomus species (s2, two ESTs). In addition, both isoforms were detected in a recent transcriptomic analysis of nodule development of M. truncatula during symbiosis with S. meliloti (El Yahyaoui et al., 2004 ) in which they were the sole Trxs that were overexpressed in this situation. To determine whether Trxs s exist in other plant species, we examined all the nucleic sequences available in databases using the BLAST program. This search did not detect any possible orthologs, even when it was extended to the rice (Oryza sativa) and poplar genomic sequences or to ESTs of the legumes Lotus japonicus and soybean.
To investigate whether the newly discovered Trxs are functional and expressed proteins, cDNAs encoding the putative mature forms of Trxs s were cloned in pRSF2, a vector designed to produce recombinant proteins in fusion with an N-terminal His-tag. The proteins were overexpressed in E. coli and purified to an apparent homogeneity in two steps consisting of an initial anion-exchange followed by an affinity chromatography on nickel-chelating Sepharose. The His-tag was then cleaved and eliminated. When subjected to SDS-PAGE (Fig. 3A ), recombinant Trx s1 (Trx s1r) and Trx s2 (Trx s2r) stained with Coomassie Blue appear as single bands with the expected molecular masses of about 13 and 14 kD, well in agreement with the theoretical masses of 13.099 and 13.998 kD calculated for the Trx s1r and Trx s2r without the transit peptide, respectively.
Then, we used either a specific peptide, in the case of Trx s1, or the recombinant protein, in the case of Trx s2, to raise polyclonal antibodies in rabbit (see "Materials and Methods"). Each antibody was purified using either the corresponding peptide or the recombinant protein immobilized on Sepharose beads before use. The specificity of the purified antibodies was tested by western blotting using recombinant proteins. Figure 3B shows that both antibodies (at a 1/1,000 dilution) were able to react with the protein against which they were raised. Amounts per lane as low as 10 ng and 3 ng could be detected with the anti-s1 and the anti-s2, respectively. Figure 3B also shows that each antibody is specific for the isoform against which it was raised and does not cross-react with the other isoform. Because Trx h isoforms were reported to be abundant in plants, particularly in organs such as seeds, roots, and nodules, we also tested the reactivity of our antibodies against several M. truncatula Trx h recombinant isoforms of groups I, II, and III (h1, h2, h4, h5, h7, h8, and h9) separately produced in our laboratory (M. Renard and F. Montrichard, unpublished data). Anti-s antibodies did not react with any of these isoforms.
As mentioned above, Trx s1 and Trx s2 have unusual putative catalytic sites when compared to the WCG/PPC sequence found in other Trxs. However, Trx s1 has a site (LCSPC) in which the two Cys, in positions with the same CXXC spacing found in other Trxs, are likely to be compatible with the formation of an intramolecular disulfide bond. Thus, this isoform may have a disulfide reductase function. In contrast, Trx s2, containing a putative catalytic site with three amino acids between the two Cys residues (WCGQNC), might not display the redox properties characteristic of classical Trxs.
To determine the possible functions of Trxs s, we performed enzymatic assays and compared the results with those obtained with a classical Trx, Trx h2 from M. truncatula. First, we assayed the disulfide reductase activity of Trx s isoforms using the test that is widely performed: the reduction of insulin. In this experiment, dithiothreitol (DTT) was used as a reducing agent for Trxs, and the electron transfer from DTT to insulin, resulting from the reaction sequence DTT
We also tested whether Trxs s could act as substrates for NTRs. M. truncatula NTRA (MtNTRA; Alkhalfioui et al., 2007
The redox midpoint potentials (Em) of both recombinant Trx s isoforms were measured using redox posing with DTT redox buffers and mBBr detection of the reduced, dithiol state (Hirasawa et al., 1999 ). The titrations gave excellent fits to the Nernst equation for a two-electron redox couple and were independent of the redox equilibration times and total DTT concentrations used, as expected for equilibrium processes. At pH 7.0, Em values of –285 ± 10 and –315 ± 10 mV were determined for Trx s1r and Trx s2r, respectively. These values are in the range of those reported for Trxs of the m type (Hirasawa et al., 1999; Collin et al., 2003) and Trxs in general. These values are significantly lower than that for the GSSG/GSH couple (–245 mV at pH 7.0), which probably explains why they are only partially reduced by GSH but are fully reduced by the thermodynamically more potent reductant, DTT (Em = –330 mV at pH 7.0). The difference of 30 mV in the Em values of the two Trx s recombinant isoforms may also explain, at least in part, why a higher concentration of DTT is required to reduce Trx s2r. It should also be pointed out that the inability of MtNTRA to catalyze the reduction of Trx s2r by NADPH cannot be attributed to the fact that it has a more negative Em value than Trx s1r, as the NADP+/NADPH couple (Em = –320 mV at pH 7.0) is capable of reducing Trx s2r in a thermodynamically favorable reaction. Thus, the absence of Trx s2r reduction in this assay must arise instead from protein/protein recognition events between this NTR and Trx s2.
To determine whether the N-terminal sequence of Trxs s could act as a signal peptide that targets the proteins to the ER, as predicted from our bioinformatic analysis, transient 35S promoter-driven expression of Trx s1 and Trx s2, C terminally fused to GFP (35S:Trx s:GFP), was performed in M. truncatula and Nicotiana benthamiana. In agro-infiltrated M. truncatula leaf epidermal cells (Fig. 6A
), Trx s1:GFP displayed a reticulated pattern very reminiscent to the ER (Yang et al., 2005
To make sure that the fluorescent proteins detected in leaves corresponded to Trxs fused to GFP and not to GFP alone, agro-infiltrated leaf samples were collected and analyzed by immunoblotting using either anti-GFP or anti-s antibodies. As anticipated, bands corresponding to the expected size (about 40 kD) for Trx s1 and Trx s2 fused to GFP were detected in all cases (Supplemental Fig. S3).
As mentioned above, ESTs and transcriptomic data suggest that expression of Trxs s seems to be linked, at least at the nucleic level, to symbiosis (El Yahyaoui et al., 2004
To investigate whether the abundance of the corresponding proteins was correlated to the transcript accumulation during symbiosis, western-blot analyses were performed using protein extracts prepared from the same organs and the specific antibodies raised against Trx s1 and Trx s2 (Fig. 3B). For 50 µg of protein extracts loaded per lane, a faint band of approximately 13 kD was visible after 30 min to 1.5 h of staining using anti-s1 (1/1,000) in nodule extracts but not in other extracts examined (Fig. 3B). Thus, Trx s1 seems to be present only in nodules in a quantity that was estimated to be around  10 ng/50 µg protein by comparison with the results obtained with pure recombinant proteins. By contrast, for 25 µg of protein extracts loaded per lane, an intense band with a molecular mass of about 14 kD was observed in both lanes of symbiotic roots and nodules after only 1 to 2 min of staining using anti-s2 (1/1,000). Thus, Trx s2 is very abundant in these organs, in amounts >50 ng/25 µg protein. It is noted that, whatever the antibody used, a band with a mass of about 15 kD was also detected in leaf extracts from symbiotic plants. Although this band is not stained in lanes corresponding to leaf extracts from uninoculated plants on the blot shown in Figure 3B, this band was often stained in other blots not shown here. This band probably results from a nonspecific reaction of a protein present in leaf extracts with anti-s antibodies. Interestingly, the molecular masses of the proteins detected in symbiotic extracts are lower than those calculated from the full-length sequences with the transit peptide, i.e. about 16 and 16.7 kD for Trx s1 and Trx s2, respectively. In fact, the masses are identical to those of the recombinant proteins alone (Fig. 3B). These results, consistent with the removal of the signal peptide in vivo, not only provide additional evidence that this novel type of Trx s is imported in the ER but also indicate that mature proteins may be soluble in this compartment, as the cleaved signal peptide is the only hydrophobic region seen in their sequences. These results, showing that during symbiosis, Trx s1 is present in nodules while Trx s2 accumulates in both roots and nodules, correlate well with those obtained from quantitative RT-PCR and suggest a regulation of the expression of the genes of Trxs s at a transcriptional level.
One isoform of Trx h was reported to be abundant in symbiotic nodules of soybean (Lee et al., 2005
We present here an overview of the Trx isoforms present in M. truncatula, highlighting that M. truncatula exhibits a novel type of Trx, with two different isoforms, in addition to all the types of Trxs previously described for Arabidopsis (Meyer et al., 2002 , 2005). These two novel isoforms indeed belong to the Trx family. This classification is supported by their primary sequences, which are similar to those of Trxs, most notably to those of Trxs m (at both protein and gene levels) and by their redox potential values that are close to those of classical Trxs. Although neither of the two isoforms is able to reduce insulin, the fact that one of them can act as a substrate for MtNTRA provides additional support for their classification as members of the Trx family. However, because of the presence of an atypical catalytic site and of a signal peptide not previously found associated with Trxs, these two isoforms clearly belong to a novel type in this family. This is the first time, to our knowledge, that Trxs containing a signal peptide are described, although the presence of Trx h in the ER had been proposed previously (Marcus et al., 1991). Because the novel isoforms were found to be associated with symbiosis, they were given the name s for symbiosis: Trx s1 and Trx s2.
No orthologs were found in the genomes of Arabidopsis, rice, and poplar. Thus, it may be possible that Trxs s are specific to legume plants. However, no orthologs were found in ESTs from L. japonicus or soybean, two other model legumes, or P. sativum. The fact that no orthologs were detected in soybean is particularly surprising given that the soybean database from The Institute for Genomic Research contains 330,436 ESTs. This could be due to the relative low representation of ESTs from symbiotic roots in this database compared to M. truncatula. Alternatively, Trxs s could be unique to galegoid legumes that, like M. truncatula but unlike soybean, make indeterminate nodules. This suggests that a type of Trx could be specific to a plant family or class. Thus, the types f, m, x, y, o, and h that are common to Arabidopsis and M. truncatula and were also found to be present in cereals (Meyer et al., 2006 In this study, we also demonstrated that this novel Trx type is associated with symbiosis. The regulation of expression of both genes during interaction of M. truncatula with S. meliloti occurs at the transcriptional level. However, Trx s isoforms show different patterns of accumulation either in nodules (Trx s1) or both roots and nodules (Trx s2), suggesting that they play different roles. Moreover, as suggested by the analysis of EST data, while Trx s1 may be specific of this symbiotic interaction, Trx s2 may also play a role in symbiosis with Glomus species. It is noted that high levels of reactive oxygen species are produced during the early step of the plant-symbiont interaction. Thus, in the case of Trx s2, one cannot rule out that it could be also functional in root tissue under other oxidative stress conditions.
We were also able to demonstrate, by transient expression of proteins in N. benthamiana or M. truncatula leaves, that Trxs s can be imported in the ER. This suggests that in symbiotic roots or/and nodules, Trxs s are primarily targeted to this compartment. However, their mechanism of retention in the ER is not known, because they do not harbor the C-terminal ER retention motif H/KDEL. It is noted that the signal peptide of Trxs s is highly conserved between the two isoforms, as is also the case for a family of small and Cys-rich proteins unique to galegoid legumes that are only expressed during nodulation (Mergaert et al., 2003
This is not the first time that Trxs have been reported to be implicated in biotic interactions. Trxs as antioxidant agents are generally thought to be involved in scavenging of the reactive oxygen species that are produced at high levels under these conditions. It may be possible that Trxs s in M. truncatula symbiotic organs play a similar role. The Trxs s may also be involved in maintaining proteins that play important roles in symbiosis in the reduced state. For this reason, it would be interesting to identify their reductase and targets. However, Trxs s may also be involved more directly in the biotic interactions. Indeed, an important role of a Trx h in self-incompatibility has been reported in Brassica (Bower et al., 1996
It is noteworthy that the Trxs of type s found in M. truncatula are clearly unique when compared with members of other types having a role in biotic interactions in that they are accumulated during symbiosis and are not synthesized under other situations. Nevertheless, Trxs s are not the sole isoforms present in symbiotic roots and nodules. They are accumulated together with one or more Trx h isoforms that may be related to the isoform reported to play a role in soybean nodulation (Lee et al., 2005
Materials Surface-sterilized seeds of Medicago truncatula (Paraggio and Jemalong A17) were imbibed on filter paper (Whatman no. 1) in a petri dish (9-cm diameter) soaked with 3.5 mL of distilled water and allowed to germinate at 20°C in the dark for 48 h. Some of the seedlings were then transferred to a mix of sand and vermiculite, and the other seedlings were transferred to soil. Plants were allowed to grow under a 16-h-light/8-h-dark photoperiod with regular watering. Symbiotic plants were obtained by inoculation of a part of 4-d-old seedlings transferred in sand with a fresh suspension of Sinorhizobium meliloti strain RCR2011 (300 µL of bacterial suspension with optical density at 600 nm of 0.1/plant). Leaves, roots, and functioning nodules (when present) were harvested after 20 to 25 d of growth. Seeds were obtained from plants that were allowed to develop in soil, generally about 3 months after the transfer of seedlings.
DNA Cloning and Overexpression of Recombinant Proteins
For cDNA cloning, total RNAs from roots of 20-d-old plants of M. truncatula Paraggio were extracted with the RNeasy plant kit (Qiagen) and reverse transcribed using the Moloney murine leukemia virus reverse transcriptase (Promega) according to the manufacturer's instructions. Complete Trx coding regions or regions corresponding to the putative mature proteins were amplified by PCR using KOD HiFi DNA polymerase (Novagen) with primers (ORFsens or Matsens and ORFanti) and cycles indicated in Table II. The PCR products were resolved in 1.4% agarose gels, and the amplicons of expected size were excised from the gels, purified with the Qiaquick gel extraction kit (Qiagen), and inserted into the plasmid pRSF2 of the ligation-independent Ek/LIC cloning kit (Novagen). This vector was engineered to express target proteins fused to a N-terminal His-tag. Recombinant plasmids were introduced in Nova Blue Escherichia coli strain for multiplication. When appropriate, they were subsequently transferred into BL21 (pLysS) cells for the production of proteins. Protein purification on Q-Sepharose and on Ni2+-chelating Sepharose (Amersham) was carried out according to the manufacturer's instructions. The His-tag was cleaved and removed using the Enterokinase cleavage-capture kit (Novagen) according the manufacturer's instructions.
Quantitative RT-PCR
For soluble protein extraction, dry or germinating seeds, leaves, and roots from 20- to 25-d-old plants grown either under sterile conditions or within symbiosis interaction with S. meliloti, and functioning nodules were ground in 50 mM Tris-HCl, pH 7.8, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride (1–5 mL/g fresh weight). The resulting homogenates were centrifuged (50,000g for 20 min at 4°C), and the soluble proteins were stored at –20°C. For western-blot analysis, proteins (25–50 µg/lane) were resolved by 15% SDS-PAGE (Laemmli, 1970 Two rabbit polyclonal antibodies were raised against a synthetic peptide (VDDNQLIPSKYGIKGIPNV) derived from Trx s1 and the recombinant Trx s2r (Davids Biotechnologie). Then they were respectively purified by affinity chromatography on the peptide and the recombinant protein bound to activated CNBr-Sepharose. Membranes were probed with 1:1,000 (v/v) dilutions of purified antibodies. Immunodetection was performed using the phosphatase alkaline assay in the presence of a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Sigma).
Trx activity was measured using the insulin or DTNB reduction assays as previously described (Holmgren, 1979 The ability of different reducing agents (DTT and GSH) or NADPH plus NTR to reduce Trxs was tested by incubating Trxs in the presence of these compounds followed by a treatment with mBBr, a fluorescent probe that labels SH. For that purpose, 100 µg of Trx in 20 mM KH2PO4 buffer, pH 7.2, was incubated in the absence or the presence of 2.5 or 20 mM DTT, 5 mM GSH, 0.2 mM NADPH alone or 0.2 mM NADPH plus 1 µM MtNTRA for 15 min, followed by addition of 5 mM mBBr. After an additional incubation period of 15 min, the proteins were precipitated by adding 5 volumes of acetone that had been prechilled at –20°C. After 2 h at –20°C, the proteins were resuspended in phosphate buffer and resolved in 15% SDS-PAGE (2 µg/lane). The fluorescent proteins were observed under UV light before the staining of proteins with Coomassie Blue.
The midpoint redox potential of each Trx s was determined according to Hirasawa et al. (1999)
Transient expression of Trxs in fusion to GFP in M. truncatula or N. benthamiana leaves was used to determine the subcellular localization of Trxs s. For that purpose, constructs were made using Gateway cloning technology (Invitrogen) according to the manufacturer's instructions. The cDNA corresponding to the two Trx s isoforms were amplified by PCR using recombinant pRSF2 plasmids with "AttB" primers (Table II) that provided for the addition of attB recombination sites and then cloned into the pDON207 entry vector using the Gateway BP Clonase enzyme mix. The constructs were checked by DNA sequencing (MWG-Biotech). For expression of the recombinant Trxs fused to GFP, the cDNAs were transferred using the Gateway LR Clonase enzyme mix into the Destination vector pK7FWG2, allowing a constitutive transcription of the gene of interest under the control of a cauliflower mosaic virus 35 S promoter (35S:Trx:GFP). These constructs were checked by restriction digest analysis. Recombinant plasmids were then transferred in Agrobacterium tumefasciens (strains GV3101 or pMP90). Finally, positive clones grown in Luria-Bertani medium supplemented with spectinomycin were resuspended in water, and suspensions with optical density of 0.2 to 0.5 at 650 nm were used to infiltrate leaves of M. truncatula or N. benthamiana. Three to 4 d later, cortical regions of epidermal cells in discs of infiltrated leaves were observed by confocal microscopy.
Protein contents in extracts were determined according to Bradford (1976)
The nucleotide sequences of Trx h2, Trx s1, and Trx s2 from M. truncatula reported in this article have been submitted to GenBank under accession numbers DQ121443, DQ121444, and DQ121445, respectively.
The following materials are available in the online version of this article.
We thank R. Tsien for mRFP1 plasmid, A. Nebenführ for ManI:Tdtomato, and V. Gomord for RFP:HDEL, Institut de Biologie Moléculaire des Plantes, Strasbourg, France. Received May 30, 2008; accepted July 3, 2008; published July 9, 2008.
1 This work was supported by the "Contrat Etat Region des Pays de la Loire" (2000–2006), by the "Conseil Général de Maine et Loire" (fellowship to F.A.), and by the Robert A. Welch Foundation (grant no. D–0710 to D.B.K.). 
2 Present address: LC1, UMR 7175, Institut Gilbert Laustriat, Pôle API, boulevard Brant, BP 10413, 67412 Illkirch cedex, France. 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.plantphysiol.org) is: Françoise Montrichard (francoise.montrichard{at}univ-angers.fr).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.123778 * Corresponding author; e-mail francoise.montrichard{at}univ-angers.fr.
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Trx
CT, using mean values obtained with two constitutive genes, Msc27 and 18S RNA (Montrichard et al., 2003
