Identification and Characterization of Thioredoxin h Isoforms Differentially Expressed in Germinating Seeds of the Model Legume Medicago truncatula^,,

Abstract

Thioredoxins (Trxs) h, small disulfide reductases, and NADP-thioredoxin reductases (NTRs) have been shown to accumulate in seeds of different plant species and play important roles in seed physiology. However, little is known about the identity, properties, and subcellular location of Trx h isoforms that are abundant in legume seeds. To fill this gap, in this work, we characterized the Trx h family of Medicago truncatula, a model legume, and then explored the activity and localization of Trx h isoforms accumulating in seeds. Twelve Trx h isoforms were identified in M. truncatula. They belong to the groups previously described: h1 to h3 (group I), h4 to h7 (group II), and h8 to h12 (group III). Isoforms of groups I and II were found to be reduced by M. truncatula NTRA, but with different efficiencies, Trxs of group II being more efficiently reduced than Trxs of group I. In contrast, their insulin disulfide-reducing activity varies greatly and independently of the group to which they belong. Furthermore, Trxs h1, h2, and h6 were found to be present in dry and germinating seeds. Trxs h1 and, to a lesser extent, h2 are abundant in both embryonic axes and cotyledons, while Trx h6 is mainly present in cotyledons. Thus, M. truncatula seeds contain distinct isoforms of Trx h that differ in spatial distribution and kinetic properties, suggesting that they play different roles. Because we show that Trx h6 is targeted to the tonoplast, the possible role of this isoform during germination is finally discussed.

Thioredoxins (Trxs) are small and powerful disulfide reductases with two close and reactive Cys residues in the conserved motif: WCG/PPC (Holmgren, 1985). In their dithiol form, they act as hydrogen donors for redox enzymes like ribonucleotide reductase, the enzyme that led to their discovery (Laurent et al., 1964). They also play a posttranslational regulatory role on protein targets involved in an ever-increasing number of cellular processes. Established redox-regulated processes in plants include carbon assimilation, seed germination, self-incompatibility reaction, redox signaling, radical scavenging, and detoxification (Buchanan and Balmer, 2005; Montrichard et al., 2009; Xie et al., 2009).

Trx isoforms constitute a particularly important protein family in plants, since 22 genes have been detected in the fully sequenced genome of Arabidopsis (Arabidopsis thaliana; Meyer et al., 2005). The family is divided into several types. The well-known f and m types as well as the more recently described and related x, y, and z types are addressed to the chloroplasts (Buchanan, 1991; Mestres-Ortega and Meyer, 1999; Lemaire et al., 2003b; Rivas et al., 2004; Arsova et al., 2010). Trxs f, m, x, and y are notably involved in the regulation of carbon metabolism and detoxification of peroxides (Collin et al., 2003, 2004; Navrot et al., 2006), while Trxs z, earlier named CITRX, are involved in the compatibility of the interaction between tomato (Solanum lycopersicum) and Cladosporium fulvum (Rivas et al., 2004) or intervene in plastid gene expression (Arsova et al., 2010). The o type is located in the mitochondria (Laloi et al., 2004), while the h type has a wide distribution, as isoforms were described to be abundant in the phloem sap and the cytosol (Ishiwatari et al., 1995; Schobert et al., 1998), anchored in the plasma membrane (Shi and Bhattacharyya, 1996), accumulated in nuclei (Serrato et al., 2001; Serrato and Cejudo, 2003), associated with mitochondria (Gelhaye et al., 2004a), or even secreted in the apoplast or the extracellular matrix (Juárez-Díaz et al., 2006; Zhang et al., 2009). Currently, the classification into most of these types is shared by dicots, monocots (Meyer et al., 2006), and green algae (Lemaire et al., 2003a), with some exceptions. Indeed, an overview of the Trx isoforms present in Medicago truncatula has recently revealed the existence of a type of Trx in this leguminous plant in addition to those mentioned above. This novel type, named s, is targeted to the endoplasmic reticulum and appears to be dedicated to symbiosis (Alkhalfioui et al., 2008). Thus, a particular type of Trx can be specific to a plant class.

Trx h isoforms constitute the largest type in the Trx family. They were further subdivided into three groups according to both their primary sequences and their activities (Juttner et al., 2000; Gelhaye et al., 2003a, 2003b, 2004b). Groups I and II of Trxs h are bona fide Trxs, being reduced by NADP-thioredoxin reductase (NTR) in the presence of NADPH. In contrast, group III includes Trx h-like isoforms with dicysteinic or monocysteinic forms (WCxxS), first described in Arabidopsis (Meyer et al., 2002) but also present in poplar (Populus spp.) and rice (Oryza sativa; Gelhaye et al., 2003a, 2004b; Meyer et al., 2005, 2006). Poplar isoforms were further shown to be reduced either by glutathione (GSH) or glutaredoxins (Grxs).

Trxs from group I and II have a protein disulfide reductase activity, while Trxs from group III have protein disulfide reductase or protein disulfide isomerase activities (Gelhaye et al., 2003a; Serrato et al., 2008).

NTRs of A/B type and Trxs h accumulate at a high level in seeds (Lozano et al., 1996; Gautier et al., 1998; Serrato et al., 2001, 2002; Maeda et al., 2003; Marx et al., 2003; Montrichard et al., 2003; Serrato and Cejudo, 2003; Cazalis et al., 2006; Alkhalfioui et al., 2007a) and play an important role in seed physiology. Indeed, Trx h isoforms were first shown to promote the activation of α-amylase, pullulanase, and proteases together with the reduction of storage proteins, resulting in the mobilization of carbohydrate and protein reserves that sustains seed germination (Kobrehel et al., 1991, 1992; Jiao et al., 1992, 1993; Wong et al., 1995; Besse et al., 1996; Lozano et al., 1996; Yano et al., 2001a). An involvement of Trxs h in reserve accumulation and storage protein structure during seed development was further proposed (Serrato and Cejudo, 2003; Guo et al., 2007). Experiments with transgenic grain have confirmed these conclusions. Trx overexpressed in barley (Hordeum vulgare) endosperm accelerated germination, the accompanying release of starch-hydrolyzing enzymes, and the reduction of storage proteins (Cho et al., 1999; Wong et al., 2002). Conversely, seeds of transgenic wheat (Triticum aestivum) in which Trx expression was suppressed showed a retardation in germination (Guo et al., 2007; Li et al., 2009). Beside this function in reserve accumulation and mobilization, Trxs h may have additional roles in seeds. Indeed, in wheat seeds, in which they show a predominant location in the nucleus of aleurone and scutellum cells, a function in the protection of tissues that suffer oxidative stress during maturation and germination was postulated (Serrato et al., 2001; Serrato and Cejudo, 2003; Pulido et al., 2009). In addition, several putative targets have been identified in cereal and leguminous seeds during development or germination (Yano et al., 2001b; Marx et al., 2003; Wong et al., 2003, 2004; Maeda et al., 2004; Balmer et al., 2006; Alkhalfioui et al., 2007b; Hägglund et al., 2008), some of them being reduced upon germination (Yano and Kuroda, 2006; Alkhalfioui et al., 2007b).

Although Trxs h were demonstrated to play important roles in seed development and germination, the exact identity of isoforms that accumulate in seeds has not been systematically investigated, and little is known about their subcellular localization. To fill this gap, in this work, we took advantage of the growing knowledge concerning the genes and genome of the model plant Medicago truncatula to identify and characterize the Trx h family of leguminous plants, to explore the activity and to determine the localization of members that are abundant in germinating seeds. Embryonic axes and cotyledons were analyzed separately because the axis is devoted to developing in shoot and roots while cotyledons are dedicated to differentiating in first leaves. This study led to the identification of three Trx h isoforms present in seeds, one of which accumulates in cotyledons. Because the latter is targeted to the tonoplast, the possible role of this isoform in seeds is finally discussed.

RESULTS

Identification of Trx h Sequences in M. truncatula Databases

Large public databases are available for M. truncatula cv Jemalong (http//www.tigr.org/tdb/mtgi; http://www.medicago.org). MtGI notably comprised 268,712 ESTs in 68,848 unique sequences at its last release (April 15, 2010), corresponding to various tissues of plants grown in either optimal or suboptimal conditions, within symbiotic interactions or not. From EST libraries, we were able to identify by homology searches about 80 sequences, tentative consensus (TC) or singletons, related to Trx, from which 11 TCs of putative Trxs h were identified (Table I). Two of them, TC153506 (89 ESTs) and TC169216 (three ESTs), are nevertheless very similar and probably correspond to a same gene, named h1 in this study, the sequence of which is still not known. They may result from sequencing errors or correspond to two slightly distinct messengers expressed from the same gene by an alternative splicing. Genes corresponding to the other nine TCs were found in genomic databases, where two additional Trx h sequences, for which no EST exists, were deposited recently, during the writing of the article. All these results allowed us to define 12 Trx h genes for M. truncatula, a number similar to that of Arabidopsis. It should be noted that two supplementary Trx sequences (TC167868 and TC169151) related to Trxs of h type were also found in MtGI. Although these sequences were deposited into the database as M. truncatula sequences, they are actually encoded by the genome of Glomus intraradices, a M. truncatula symbiont (D. Van Thuinen and F. Montrichard, unpublished data).

Table I. Twelve isoforms of Trx h were found in M. truncatula Jemalong databases.

The group to which they belong, the name they were given in this work, and the corresponding numbers of ESTs, TC, bacterial artificial chromosome (BAC), and chromosome are indicated.

Group	Name	No. of ESTs	TC	BAC	Chromosome
I	h1	89	TC153506	–	–
		3	TC169216
	h2	20	TC154689	AC160013_5	1
	h3	0	–	CR955005_31	5
II	h4	34	TC146559	AC151524_26	7
	h5	2	TC165299	CR954189_16	5
	h6	3	TC158033	CR954189_24	5
	h7	4	TC155044	CR954189_15	5
III	h8	12	TC146257	AC152552_10	7
	h9	34	TC146438	AC169075_9	2
	h10	9	TC147788	AC231337_5	2
	h11	13	TC152067	AC144502_13	4
	h12	0	–	AC174331_38	8
				AC151949_28

The names given to the 12 isoforms of Trx h found in M. truncatula (Mth1–Mth12) as well as the corresponding numbers of ESTs and of TCs, bacterial artificial chromosomes, and chromosomes are indicated in Table I. The proteins are aligned in Figure 1, and predictions of their localization, using software available on the Expasy site (http://www.expasy.ch), are provided in Supplemental Table S1. Their sequences are finally compared in a phylogenic tree with those known for Trxs h in Arabidopsis, in pea (Pisum sativum), a crop species related to M. truncatula, and in cereal seeds (Fig. 2). This tree shows that all Trxs h from M. truncatula are members of the groups already defined for Trxs h from Arabidopsis and poplar. Three belong to group I, four to group II, and five to group III. It is noted that all Trxs h identified in cereal seeds belong to group I.

Figure 1.

Alignment of Trxs h from M. truncatula Jemalong. Protein sequences were deduced from nucleic acid sequences found in databases (Table I). The accession numbers in GenBank for encoded Trx h1, h2, h4, h5, h6, h7, h8, and h9 are AAZ98842.1, AAZ98843.1, ACZ37068.1, ACZ37069.1, ACZ37070.1, ACZ37071.1, ACZ37072.1, and ACZ37073.1, respectively. Identical and conserved amino acid residues appear in red and blue, respectively. Residues that are identical in several sequences but not all appear in green. The sequence corresponding to the catalytic site is in boldface.

Figure 2.

Phylogenic tree of M. truncatula, pea, Arabidopsis, and cereal Trxs h. Proteins were deduced from nucleic acid sequences of M. truncatula Jemalong (Table I); pea Psh1 (AJ319808), Psh2 (AJ310990), Psh3 (AY170650), and Psh4 (AY170651); Arabidopsis Ath1 (AT3G51030), Ath2 (AT5G39950), Ath3 (AT5G42980), Ath4 (AT1G19730), Ath5 (AT1G45145), Ath7 (AT1G59730), Ath8 (AT1G69880), Ath9 (AT3G08710), Ath10 (AT3G56420), AtCxxS1 (AT2G40790), and AtCxxS2 (AT1G11530); barley Hvh1 (AY27300) and Hvh2 (ABX09990); T. aestivum Capitole Ta (X69915); T. aestivum Chinese Spring TahA (AJ009762) and TahB (AJ404845); T. aestivum Soisson Tah1 (AY072771), Tah2 (AF286593), and Tah3 (AF420472); and T. durum Td (AJ00903). Protein sequences were aligned with ClustalW (default parameters), and an “unrooted neighbor-joining” tree was constructed (http://clustalw.genome.jp/).

Mth1 to Mth7 of group I and II exhibit the classical catalytic site: WCGPC (Fig. 1). A particularity in the sequence of some Trxs h of group I in Arabidopsis and poplar is the presence of a variant catalytic site: WCPPC. Three out of four Trxs of this group notably have this atypical site in Arabidopsis. In contrast, none of the Trxs h of group I identified in M. truncatula (h1–h3; Fig. 1) or known in pea (Montrichard et al., 2003; Traverso et al., 2007) has such a site. Isoforms of group I of M. truncatula have no predicted targeting sequence and may be cytosolic (Supplemental Table S1). However, Mth1 and Mth2 exhibit an N-terminal motif, MAAEE, that was first described for a well-studied Trx h in rice, early found in the phloem sap, and suggested to be involved in its cell-to-cell transport and phloem trafficking through plasmodesmata (Ishiwatari et al., 1998). In a recent study, this rice isoform was further shown to be secreted in the apoplastic compartment of root cells in response to cold stress (Zhang et al., 2009). Therefore, Mth1 and Mth2 of M. truncatula may also be secreted in the phloem or the apoplast, while h3 (if synthesized; see below), which does not possess this N-terminal motif, may only be restricted to the cytosol.

Trxs of group II differ from Trxs of group I by the presence of an N-terminal extension. Although the available algorithms do not generally predict any obvious targeting signal, these extensions may be involved in the final location of the proteins, which varies greatly from one isoform to another. Indeed, isoforms were shown to be addressed to mitochondria in poplar (Gelhaye et al., 2004a) or to the stylar extracellular matrix in Nicotiana alata (Juárez-Díaz et al., 2006). In leguminous plants such as Glycine max, two Trxs h from this group were earlier described to be anchored in the plasma membrane (Shi and Bhattacharyya, 1996), while another isoform was recently shown to be associated with nodules of plants grown in symbiosis with Sinorhizobium melilotii (Lee et al., 2005). In M. truncatula, this group includes isoforms h4 to h7 (Fig. 2). Mth5 to Mth7 share highly similar sequences (Fig. 1) and belong to the same cluster that is different from that of Mth4 (Fig. 2). Because their genes are located on the same chromosome (chromosome 5), they most probably arose from two recent duplication events. Mth4 is predicted to be addressed to plastids (Supplemental Table S1). However, because no Trx h has been demonstrated to be present in this compartment so far and peptides addressing proteins to plastids and mitochondria have similarities, Mth4 may be the ortholog of Pth2, the poplar isoform mentioned above and located to the mitochondria. According to predictions, Mth5 and Mth7 may accumulate in the cytosol, while Mth6 is possibly targeted to the cytoskeleton (Supplemental Table S1). To our knowledge, this is the first time that a cytoskeleton localization has been proposed for a Trx. Mth4, Mth6, and Mth7, which have a conserved Gly residue at position 2, are also predicted to be myristoylated (with score values of 0.95–1; Myristoylator on the Expasy site).

In group III, isoforms from M. truncatula (h8–h12) are either monocysteinic or dicysteinic (Fig. 1), like those from Arabidopsis (Meyer et al., 2002), poplar (Gelhaye et al., 2003b), and rice (Meyer et al., 2005). Only dicysteinic forms, Mth8 and Mth12, have an N-terminal extension. This extension is slightly longer for h12 than for h8, the extension of which is similar to that of Trxs of group II (Fig. 1). Mth8 is predicted to be myristoylated (score of 1) and to be targeted to plastids (Supplemental Table S1). However, dicysteinic forms of Trxs h of group III are highly conserved among species (Juttner et al., 2000; Gelhaye et al., 2003a; Koh et al., 2008), and the possible ortholog of Mth8 in Arabidopsis, Ath9 (Supplemental Fig. S1), has recently been demonstrated to be associated with plasma membrane, possibly via myristoylation (Meng et al., 2010). Thus, Mth8 may also be associated with plasma membrane through the same mechanism. Regarding the localization of Mth12, prediction is less clear (Supplemental Table S1). Monocysteinic Mth9 to Mth11, with a size similar to that of Trxs h of group I, also have no obvious predicted localization (Supplemental Table S1).

Cloning and Overexpression of M. truncatula Trx h Isoforms in Escherichia coli

To investigate whether the Trx h sequences encode functional proteins, the coding regions of selected isoforms were amplified by reverse transcription-PCR, starting with RNAs extracted from 14-h-imbibed but not germinated seeds of M. truncatula cv Paraggio and cloned into the expression vector pRSF2. This vector was designed to allow the production of recombinant proteins in fusion with an N-terminal His tag. It is noted that Jemalong is the model genotype used in M. truncatula databases. This genotype was here used to construct Table I, Supplemental Table S1, and Figures 1 and 2, whereas the Paraggio genotype was used for the rest of the study.

Most classical Trx isoforms of groups I (h1 and h2) and II (h4–h7) were cloned from Paraggio. Mth3 of group I, whose genomic sequence appeared in the data bank during the writing of this article, was not studied, the status of this gene as an expressed gene being uncertain. Indeed, no corresponding EST can be found in data banks, suggesting that the gene is not functional. Interestingly, the possible ortholog in pea (Psh2; Fig. 2) does not seem to be transcribed either (Traverso et al., 2007). Another possibility is that these ortholog genes are expressed in an organ or a situation not examined so far. Dicysteinic Mth8 and monocysteinic Mth9 of group III were also cloned for comparison. Paraggio sequences were determined and deposited in GenBank with the accession numbers indicated in Table II. They were compared with those of Jemalong. Only a few differences were detected in protein sequences between orthologs originating from the two genotypes. They are listed in Table II. From the recombinant Trxs overexpressed in E. coli, all were found to be highly expressed as soluble proteins, except h6, which was recovered in inclusion bodies, a behavior rather unusual for classical Trxs. This Trx can have a limited solubility due to its peculiar N-terminal sequence, which possibly targets the isoform to the cytoskeleton. Several attempts to solubilize the full-length protein under a stable form, notably using urea, were unsuccessful, the protein aggregating after the removal of the solubilizing agents by dialysis. Because the removal of the transit peptide often improves the solubility of recombinant proteins, we cloned the coding region of h6 without its 5′ end sequence. We used the protein alignment shown in Figure 1 to determine which amino acids to remove. A truncated h6 protein, deleted from the first 13 amino acids, was then overexpressed in E. coli and found to be both soluble and stable. Finally, all recombinant Trxs h were purified using two chromatography steps on ion exchanger and then Ni²⁺-chelating Sepharose.

Table II. Accession numbers and biochemical properties of Trxs h from M. truncatula Paraggio.

The differences of ortholog sequences in Paraggio and Jemalong are indicated in the right column.

Name	Accession No.	No. of Amino Acids	Molecular Mass	pI	Paraggio/Jemalong
			D
h1	DQ121442	117	12,798	5.61	D-103/E-103
h2	DQ121443	120	13,338	4.99
h4	FJ858140	131	14,537	5.76	V-8G-12P-13/L-8D-12S-13; insertion: A-14T-15A-16
h5	FJ858141	126	14,598	9.60
h6	FJ858142	128	14,529	9.40
	(without N terminus)	(115)	(13,294)	(9.56)
h7	FJ858143	127	14,380	9.85	E-85/D-85
h8	FJ858144	139	15,589	4.63
h9	FJ858145	124	13,884	4.40

For an SDS-PAGE analysis, the His tag was cleaved and eliminated. After electrophoresis, recombinant Trxs h stained with Coomassie Brilliant Blue appear as single bands with molecular masses of about 13 to 15 kD (Fig. 3), well in agreement with their theoretical masses (Table II), except for h5, whose apparent size is slightly lower than expected. Differences between theoretical and apparent masses can occur with small proteins like Trxs and were also observed for recombinant Trxs originating from barley (Maeda et al., 2003). Antibodies raised against Psh3 and Psh4 from pea (Montrichard et al., 2003) were tested for cross-reaction with M. truncatula h isoforms in western blotting. Figure 3 shows that all the isoforms were detected with a mix of these antibodies, with an efficiency that varies from one isoform to another: h1 > h4 = h7 > h6 > h2 = h5. In contrast, the two isoforms of group III (h8 and h9) were not detected by these antibodies.

Figure 3.

Analysis of purified recombinant Trxs h by SDS-PAGE and western blotting. Recombinant Trxs h were overexpressed in fusion with a His tag in E. coli and purified. After purification and removal of the His tag, they were resolved by 15% (w/v) SDS-PAGE and stained with Coomassie Brilliant Blue (top panel) or transferred onto nitrocellulose membranes. Blots were probed using the alkaline phosphatase assay with a mix of antibodies raised against pea Trxs h3 and h4 (Montrichard et al., 2003) used at a 1:500 (v/v) dilution (bottom panel). For SDS-PAGE and western-blot analyses, 1 μg and 100 ng of each recombinant protein were loaded per lane, respectively. Molecular masses of standard proteins are indicated in kD at the left.

Functional Study of Recombinant Trx h Isoforms

To compare the activities of the different Trx h isoforms from M. truncatula, we performed a functional study using the purified recombinant Trxs h and NTRA from M. truncatula (MtNTRA). This enzyme was previously characterized and shown to be present in seeds, axes, and cotyledons (Alkhalfioui et al., 2007a). MtNTRA is most probably the ortholog of AtNTRA, reported to target both the cytoplasm and mitochondria of Arabidopsis cells (Reichheld et al., 2005). In the NADP-thioredoxin system (NTS), electrons flow from NADPH to Trx h, which in turn reduces its target according to the following equation: NADPH → NTR → Trx h → target. The flux of electrons may then vary with the ability of NTR to reduce a given Trx h isoform as well as with the level of reductase activity of the given Trx h isoform on its target. Thus, we first tested the capacity of NTRA to reduce Trx h isoforms using dithiobisnitrobenzoic acid (DTNB) as a final electron acceptor. As expected, NTRA was found to be active only on isoforms of group I and II (Table III). The K_m values calculated are in the range of those determined for NTR and Trxs h from Arabidopsis, poplar, wheat, and barley (i.e. 0.7–20 μm; Rivera-Madrid et al., 1995; Gelhaye et al., 2002; Serrato et al., 2002; Shahpiri et al., 2008). However, they vary greatly with the isoform considered. Indeed, NTRA exhibits a lower affinity for isoforms of group I (K_m ≥ 10 μm) than for isoforms of group II (K_m around 1–2 μm), except for h6, for which a K_m of 6.8 μm was determined. Noteworthy, by contrast to the other recombinant Trxs h overexpressed in this study, h6 was produced as a truncated protein without its first 13 amino acids. The absence of the N-terminal sequence may have an effect on the structure of h6 and its capacity to interact with NTRA. The k_cat values were also calculated (Table III). The values were similar for isoforms of the two groups, ranging from 0.87 to 2.11 mole of Trx reduced per second per mole of NTRA. Consequently, the efficiency of NTRA (k_cat/K_m) is lower with Trxs h of group I than with Trxs h of group II. NTRA is notably 25-fold less efficient with h1 than with h7.

Table III. Kinetic parameters of NTR activity in the presence of different recombinant Trxs h (DTNB assay).

Parameter	h1	h2	h4	h5	h6	h7	h8	h9
Group	I	I	II	II	II	II	III	III
Km (μm)	10.1 ± 0.3	13.1 ± 1.1	1.6 ± 0.3	2.4 ± 0.2	6.8 ± 0.2	0.8 ± 0.1	–	–
kcat (s−1)	0.87 ± 0.01	1.47 ± 0.05	1.84 ± 0.30	2.05 ± 0.08	2.11 ± 0.27	2.08 ± 0.10	–	–
kcat/Km (s−1 μm−1)	0.086	0.112	1.146	0.855	0.313	2.218	–	–

We then tested the disulfide reductase activity of recombinant Trxs h using the test that is the most widely used: the reduction of insulin in the presence of dithiothreitol (DTT), a potent reducing agent for Trxs. It should be noted that Trx-like proteins and Grxs can also be reduced by DTT and be active in this test. Isoforms from group I and II were found to be able to reduce insulin that, once reduced, precipitated (Supplemental Fig. S2). However, the rate of insulin precipitation differs greatly with the isoform considered, Mth4 and Mth2 being the most efficient, Mth1, Mth6, and Mth7 having an intermediary efficiency, and Mth5 being poorly efficient (Fig. 4). In group III, Mth8 was found to present a limited action on insulin precipitation that is similar to that of Mth5, while Mth9 has no effect (Supplemental Figs. S2 and S4). Thus, the rate of insulin reduction varies greatly and independently of the group to which the isoforms belong: h4 > h2 > h6 ≥ h1 ≥ h7 > h5 = h8 > h9 = 0. It is noteworthy that Mth8 is not reduced by MtNTRA (Table III). To test whether it can be reduced by GSH, we replaced DTT by GSH in the insulin test. A reduction of insulin was indeed observed in the presence of 5 and 10 mm GSH, although the rate of reduction was 2-fold lower with 10 mm GSH than with 1 mm DTT (Supplemental Fig. S3). This figure nevertheless shows that the rate was highly increased when a Grx was added to the reaction mixture containing GSH (Supplemental Fig. S3).

Figure 4.

Insulin reduction by recombinant Trxs h. The disulfide reductase activity of Trxs h was assayed by measuring the rate of precipitation of insulin upon reduction, which was followed by turbidimetry at 650 nm (for detailed experimental conditions, see Supplemental Fig. S2). OD, Optical density.

Trx h Abundance in Dry and Germinating Seeds of M. truncatula

Previous studies performed with pea and barley seeds have shown no correlation between Trx h mRNA abundance and protein accumulation (Montrichard et al., 2003; Shahpiri et al., 2008). This prompted us to analyze the Trx h gene expression in M. truncatula seeds only at the protein level, using the anti-h antibodies mentioned above. However, it is generally difficult to determine both the number and the nature of isoforms present in an extract in one-dimensional (1D) western blotting, because Trxs h have similar masses and antibodies raised against one isoform often cross react with other isoforms of the same species (Lozano et al., 1996; Serrato et al., 2001; Marx et al., 2003) or of related species (Fig. 3). Thus, to determine the number and the identity of Trx isoforms present in seeds, we performed the analysis by two-dimensional (2D) western blotting. In these conditions, Trxs that have different pI values (Table II) could be separated.

A few dots were detected on 2D blots corresponding to seed extracts that were probed with anti-h antibodies (Fig. 5). This figure shows that the pattern is almost the same whatever the time of seed imbibition, 0 h (dry seeds), 14 h (before radicle protrusion), and 22 h (after radicle protrusion), whereas it differs slightly between embryo axes and cotyledons. Indeed, when considering the major signals (arrows), two dots with pI around 5 to 6, also visible on membrane corresponding to leaves or roots, and one dot in the acidic area seem to be common to the two parts of the seed, while one intense dot was only detected in the basic area of cotyledon blots. According to theoretical pI values deduced from the full-length sequences (Table II), the dots detected at pH 5 to 6 could correspond to isoforms h1, h2, and h4, whereas those detected in the basic area could correspond to h5, h6, and h7. Although the dot observed in the acidic area cannot be due to a native Trx isoform, it could correspond to an isoform that has been modified in vivo or during the experiment, a modification leading to a decrease in pI and a shift of the protein to the acidic area. The modification is probably due to an oxidation. Indeed, Trxs have highly reactive Cys residues, whose thiol group can easily be oxidized in acids.

Figure 5.

Abundance of Trxs h in seeds, roots, and leaves. Soluble proteins from embryo axes (A) and cotyledons (C) of dry seeds (0h) or seeds imbibed for 14 h (14h) or 22 h (22h) as well as proteins from roots (R) and leaves (L) from 21-d-old plants were resolved on 2D gels. Then, proteins were stained on the gels with Coomassie Brilliant Blue or transferred onto nitrocellulose membranes that were probed with anti-h antibodies. The area corresponding to small proteins with masses ranging from 10 to 15 kD (in a nonlinear gradient of pH from 3 to 10) is shown. A section of the C14h gel is also enlarged at the right.

To identify the proteins that react with anti-h antibodies, the dots were used to localize the corresponding protein spots on 2D gels stained with colloidal Coomassie Brilliant Blue. In all cases, the dots matched with minor spots of proteins, the spot corresponding to the signal detected in the basic area being notably hardly detectable on the gel stained in blue (Fig. 5). Spots were punched out and submitted to liquid chromatography-tandem mass spectrometry analysis. Mth1 was identified in both spots in the pH 5 to 6 area corresponding to axes or cotyledons from dry and imbibed seeds (Supplemental Table S2). This isoform, therefore, is present under two forms slightly differing by their pI. Mth2 was also found in mix with Mth1 in the left spot of axes and cotyledons from dry seeds. The other spots analyzed gave either no result or proteins different from Trx. Because the two spots detected in the pH 5 to 6 area were also present in leaves and roots (Fig. 5), Mth1 and Mth2 may also be present in these organs.

To ascertain the content of Trxs h in seeds, we further analyzed major proteins of 10 to 15 kD present on 2D gels corresponding to a C14 extract by mass spectrometry. We chose to restrict our analysis to this extract because no difference was observed on the blots during the time course of imbibition, and the pattern of proteins detected on cotyledon blots seemed to include those detected on axis blots. Interestingly, at the left of the area of Mth1 and Mth2, Mth6 was also found to be present. The position of Mth6 on the gel indicates that the protein is modified. One can imagine that a small proportion of the protein remains nevertheless under its native form and is responsible for the signal detected on the basic area of cotyledon 2D blots. Similarly, a modified form of Mth1 was recovered on the extreme acidic side of the gel. The presence of modified forms of Trxs h in cotyledons from 14-h-imbibed seeds is probably linked to the oxidative conditions that reign during germination (Bailly, 2004) and lead to Trx oxidation. Consistent with this hypothesis, proteins were found to be mostly oxidized in early-imbibed M. truncatula seeds (Alkhalfioui et al., 2007b).

The results described here further show that modified forms of Trxs may be much less immunoreactive than native forms, since modified Mth1 and Mth6 found in the proteome were not detected on 2D blots. Modified forms are nevertheless sufficiently abundant to be identified on 2D gels. Therefore, the two methods appeared as complementary: the native forms being detected on western blots and the modified forms being identified in the proteome. Altogether, these results show that in dry and germinating seeds, Mth1 and, to a lesser extent, Mth2 are abundant in both axes and cotyledons while Mth6 is mainly present in cotyledons.

In data banks of the model genotype Jemalong, 92, 20, and three ESTs corresponding to h1, h2, and h6 genes, respectively, have been recovered (Table I), the h1 gene being the most active Trx h gene in this genotype. ESTs corresponding to h1 and h2 were recovered in different tissues and at different stages of development, indicating rather constitutive functions for the translated proteins. By contrast, the h6 gene is one of the two lesser active Trx h genes (Table I), ESTs corresponding to this isoform being associated with developing seeds, developing leaves, and roots infected with Phytophthora medicaginis. This suggests that the encoded proteins have restricted functions, one of which seems to be dedicated to seed cotyledons.

Subcellular Localization of Trx h6

Mth6, which mainly accumulates in cotyledons of dry and germinating seeds, may have a peculiar role during germination. It belongs to the second group of Trxs h that have an N-terminal extension possibly involved in their final localization, h6 being notably predicted to be myristoylated and associated with the cytoskeleton. To determine whether the N-terminal sequence of Mth6 could act as a targeting peptide, transient 35S promoter-driven expression of Mth6, C-terminally fused to GFP (35S:Trx:GFP) or red fluorescent protein (35S:Trx:RFP), was performed in Nicotiana benthamiana and M. truncatula. In agroinfiltrated N. benthamiana leaf epidermal cells (Fig. 6A), GFP fluorescence was found to be associated with a membrane surrounding the cells, most probably the tonoplast, and with the nucleus of certain cells. Chloroplasts (Fig. 6B) and contour (Fig. 6C) of the cells observed in Figure 6A are also shown.

Figure 6.

Visualization of Trx h6 fused to fluorescent proteins (GFP or RFP) in N. benthamiana or M. truncatula leaf epidermal cells. A to D, Membrane-associated fluorescence pattern resulting from Trx h6:GFP expression in N. benthamiana. B, Autofluorescence of chloroplasts in the red channel. C, Autofluorescence of chloroplasts in the differential interference contrast channel. D, Overlay of A to C. E to K, Coexpression in M. truncatula turgescent (E–G) or plasmolyzed (H–K) leaf cells of the tonoplast marker TIP1-1:GFP (E and H) and Trx h6:RFP (F and I). Near-perfect colocalization of coexpressed proteins is observed (G and K). J shows a differential interference contrast image of the plasmolyzed cell highlighting the outline of the cell wall. Bars = 10 μm.

To make sure that the fluorescent proteins detected in leaves corresponded to h6 fused to GFP and not to GFP alone, agroinfiltrated leaf samples were collected and analyzed by immunoblotting using either anti-GFP or anti-h antibodies. As anticipated, a band corresponding to the expected size (about 40 kD) for h6 fused to GFP was detected in both cases (Supplemental Fig. S4). However, free GFP and free h6 were also detected in the lanes. The band corresponding to free GFP being much more intense than that of the fusion protein indicates that a large proportion of the fusion protein was hydrolyzed in leaf cells. Thus, the fluorescence detected in the nucleus of certain cells was most probably due to the free GFP, while that associated with membranes was likely due to the whole fusion protein. The same pattern of fluorescence was observed in M. truncatula leaves with h6:RFP. The tonoplast localization of h6 was confirmed upon coexpression of h6:RFP with TIP1-1:GFP, a protein demonstrated to be associated with the tonoplast (Boursiac et al., 2005). Nearly perfect colocalization between TIP1-1:GFP (Fig. 6E) and h6:RFP (Fig. 6F) was observed in the merged image (Fig. 6G), suggesting that h6 is associated with the tonoplast. This localization was further confirmed under plasmolysis conditions, where h6:RFP kept colocalized with TIP1-1:GFP (Fig. 6K). This is the first description, to our knowledge, of a Trx associated with the tonoplast.

DISCUSSION

The NTS has been mainly studied in germinating seeds from cereals, where its role in the mobilization of reserves was clearly established. In contrast, there is still scant information on NTS in seeds of dicotyledonous plants. Here, using data available for the model plant M. truncatula, we further characterized the Trx h family of leguminous plants and explored its role in seeds during germination.

Twelve isoforms of Trx h, a number similar to that reported for Arabidopsis and rice (Meyer et al., 2005), were found in M. truncatula databases. They belong to the three groups previously described for Arabidopsis and poplar (Meyer et al., 2002, 2005; Gelhaye et al., 2004b): three isoforms in group I, four in group II, and five in group III. Six isoforms of groups I and II out of seven and two isoforms from group III were further successfully produced as recombinant proteins.

Recombinant proteins were then used to perform a functional study with MtNTRA (Alkhalfioui et al., 2007a). This allowed the investigation of interaction between NTR and Trxs h from the same leguminous plant. The results obtained in the functional study confirmed that Trxs h from groups I and II are true Trxs h, being reduced by NTR and in turn able to reduce insulin. However, the detailed study of activity performed here showed that NTR is more efficient to reduce Trxs h from group II than those of group I. This introduces a kinetic control on the system depending on the nature and the concentration of Trx isoforms present. In the case of accumulation of isoforms from groups I and II, one can imagine that a competition between isoforms of the two groups may take place. However, they may not belong to the same cell compartments. By contrast, the rate of reduction of insulin, used here as a Trx target, varies greatly and independent of the group to which the isoforms belong: h4 >h2 > h6 ≥ h1 ≥ h7 >> h5. Thus, the flux of electrons in the NTS in vivo may also vary with the nature of the targets present.

Regarding Trxs h of group III, they are not reduced by NTR and are rather Trx-like proteins. By contrast, the dicysteinic Mth8 can be reduced by GSH, either directly or indirectly through Grxs that highly enhance the rate of reduction. These results are in accordance with those reported for their orthologs in Arabidopsis and poplar (Gelhaye et al., 2003a; Serrato et al., 2008), highlighting the interconnection of Trx and Grx systems as demonstrated recently with a mutant deficient in NTR (Reichheld et al., 2007).

Once the family of Trxs h of M. truncatula was characterized, members present in seeds were identified after separation of proteins on 2D gels. Two isoforms of group I (h1 and h2) and one isoform of group II (h6) were demonstrated to be present in dry and germinating seeds from M. truncatula. Mth6 seems to be mainly associated with cotyledons, while Mth1 and Mth2 are present in both axes and cotyledons and also probably are synthesized in leaves and roots. It appeared that Trxs can be present under modified forms in seeds, resulting both in a shift of the spots to the acidic area of the 2D gels and a decrease in their immunoreactivity. Thereby, a large part of Mth1 and Mth6 that were found as modified forms on 2D gels were not detected by western blotting. Thus, 2D western blotting was proven to be insufficient to identify all the isoforms present in seeds or to estimate their level of accumulation. The three isoforms found in seeds not only differ by their spatial distribution but also by their cellular location. Mth1 and Mth2 are most probably located in the cytosol or secreted (in the apoplast of seed cells or into the phloem of differentiated organs), as demonstrated for their orthologs in rice (Ishiwatari et al., 1998; Zhang et al., 2009) and also supposed for their ortholog in pea, Psh1, which was found to be associated with vascular tissues (Traverso et al., 2007). Besides this probable location for Mth1 and Mth2, we provide evidence of an unexpected location for Mth6, which appears, from our transient expression experiment, to be associated with the tonoplast. The binding of h6 to the tonoplast may be mediated through the myristoylation of its Gly-2 residue.

In previous studies performed with pea, three different isoforms of Trx h were also found to accumulate in seeds (Montrichard et al., 2003; Traverso et al., 2007), two isoforms of group I (Psh1 and Psh3) and one isoform of group II, Psh4. Psh1 and Psh3 are most probably the respective orthologs of Mth2 and Mth1 (Fig. 2), and, similar to them, they constitutively accumulate in all the organs examined: embryo axes, cotyledons, roots, and leaves. Thus, isoforms of group I seem to have a large distribution in legume tissues, Psh3 and Mth1 being the most abundant isoforms in seeds of pea and M. truncatula, respectively.

However, regarding the isoforms of group II, Psh4 of pea is not the ortholog of Mth6 found in M. truncatula (Fig. 2). Although the synthesis of these two isoforms seems to be restricted to seeds, Psh4 was only detected in axes of dry and imbibed but not germinated pea seeds (Montrichard et al., 2003), while Mth6 was found to mainly accumulate in cotyledons of dry and germinating M. truncatula seeds. Nevertheless, Psh4 was found in a very low quantity in pea axis. Thus, the possible ortholog of this isoform (Mth4; Fig. 2) may also be present in M. truncatula seeds, but in a quantity below the detection threshold. Similarly, an ortholog of Mth6 may exist in pea seeds but in a too-low quantity to be detected. Alternatively, the pattern of Trx h abundance in seeds differs between the two species, and this is linked to their difference in development. Indeed, pea has an epigeous growth and cotyledons die rapidly in the ground after germination, while M. truncatula has a hypogeous growth, the cotyledons differentiating in the first leaves of the established plant.

A few isoforms of Trx h were also found to accumulate in seeds of cereals. However, in contrast to pea and M. truncatula seeds, all the isoforms found in cereal seeds belong to group I (Fig. 2). In PCR experiments, one isoform was identified in Triticum durum (Gautier et al., 1998) while one to three isoforms were shown to be present in T. aestivum (wheat), depending on the cultivar analyzed: Ta in Capitole (Gautier et al., 1998), TahA and TahB in Chinese Spring (Serrato et al., 2001; Serrato and Cejudo, 2003), and Tah1, Tah2 (identical to TahB), and Tah3 in Soisson (Cazalis et al., 2006). In T. aestivum, Ta, TahA, TahB/h2, and Tah3 are highly identical (over 90%), a result that is in contrast to that obtained with Trxs h of model species that are more divergent. However, because T. aestivum is hexaploid, the finding of almost identical Trxs h may indicate that these are encoded by homolog genes originating from the three genomes. In Soisson, Tah1, with only 51% identity with Tah2 and Tah3, may nevertheless be encoded by a distinct gene. By a proteomic approach, two different isoforms of Trx h, Hvh1 and Hvh2, were detected in dry seeds of barley (Maeda et al., 2003; Shahpiri et al., 2008), a diploid cereal. Hvh1 is nearly identical to Tah1 and Hvh2 is highly similar to Ta, TahA, TahB/h2, Tah3, and Td (Fig. 2). The two isoforms of barley vary in distribution in the different seed tissues, Hvh1 being present in endosperm, aleurone layer, and embryo while Hvh2 mainly accumulates in the embryo. Interestingly, Hvh1 and Tah1 harbor the MAAEE motif and may be cytosolic or secreted, whereas Hvh2 and its wheat orthologs have an N-terminal sequence rich in Ala that possibly anchors the proteins in membranes (Gautier et al., 1998; Serrato et al., 2001). Thus, similar to isoforms found in M. truncatula seeds, Trxs h present in cereal seeds may be cytosolic and/or secreted and membrane bound.

It is noted that no Trx h of group III or plastidial Trx of f and m types were recovered during the proteome analysis of M. truncatula (this work) or barley (Maeda et al., 2003; Bønsager et al., 2007). Orthologs of these proteins were nevertheless either shown to have a role in germination or to be present in seeds of other species (Balmer et al., 2006; de Dios Barajas-López et al., 2007; Li et al., 2009; Meng et al., 2010). This indicates that these proteins are much less abundant than Trxs h identified in seeds.

The isoforms of Trx h found in M. truncatula seeds have different biochemical properties, subcellular localization, and spatial distribution; therefore, they may serve different physiological functions. During germination of cereal seeds, reduction of Trxs h was earlier shown to promote reserve mobilization and metabolism resumption (Kobrehel et al., 1991, 1992; Jiao et al., 1992, 1993; Wong et al., 1995; Besse et al., 1996; Lozano et al., 1996; Yano et al., 2001a). Such a role for Trxs during the germination of legume seeds was also evident from Trx-linked redox changes observed in germinating seeds of M. truncatula. Indeed, more than 100 Trx-linked proteins were identified in seeds; most of them that are oxidized or partly reduced in dry seeds became more reduced upon germination (Alkhalfioui et al., 2007b). In M. truncatula, h1 and h2, which are probably mostly cytosolic, can serve these functions. Mth6, which is associated with the tonoplast of cotyledon cells, may also play an important role in the reduction of the proteins stored in storage vacuoles, particularly abundant in cotyledons. This isoform is also expected to have specific targets linked to its peculiar location. It may exert a redox regulation of function of membrane proteins such as the vacuolar ATPase, a protein we identified among the potential Trx targets in seed cotyledons (Alkhalfioui et al., 2007b). Thereby, a role in water uptake through the activation of the ATPase during imbibition can be envisaged for this isoform.

Abundant cytosolic Mth1 and, to a lesser extent, Mth2 may also intervene in scavenging of reactive oxygen species (ROS) that are produced at high rates when metabolism resumes upon imbibition (Bailly, 2004). Indeed, their ortholog in pea, Psh1, was found to confer, by functional complementation, tolerance to hydrogen peroxide and the ability to reduce Met sulfoxide to a yeast mutant devoid of cytosolic Trxs (Traverso et al., 2007). These results suggest that Mth1 and Mth2 can act as substrates for detoxifying enzymes, such as peroxiredoxins that eliminate peroxides, as well as for repairing enzymes, such as Met sulfoxide reductases that reduce protein oxidized at the level of Met. Several peroxiredoxins were indeed found as potential Trx targets in seeds of M. truncatula (Alkhalfioui et al., 2007b). In wheat, Trxs h were found to be concentrated in the nucleus of aleurone and scutellum cells during germination (Serrato et al., 2001). Because these cells suffer oxidative conditions, a role in the protection of nuclear components against ROS has been proposed. In M. truncatula seeds, a fraction of cytosolic h1 and/or h2 may also be transferred to the nucleus to play this role.

Knowledge generally lags behind on the individual roles played by Trx isoforms when they coexist in the same compartment. Early experiments with complementation of the mutant of yeast devoid of cytosolic Trxs, already mentioned above, suggested a specificity of function for the five different Trxs h from Arabidopsis tested (Mouaheb et al., 1998). The in vivo specificity of Trx isoforms toward a few well-known targets was further confirmed by two-hybrid experiments (Vignols et al., 2005). Whether Mth1 and Mth2 have redundant functions and/or act on different protein targets in M. truncatula seeds remains to be determined. It would be interesting to analyze their specificity toward some of the potential targets we identified in this organ (Alkhalfioui et al., 2007b). It would also be of interest to identify the targets of Mth6 that might be rather specific, linked to the unusual location of this isoform on the tonoplast.

It is noted that Mth6 belongs to a cluster of very close isoforms in M. truncatula (h5, h6, and h7), h7 having a Gly residue at position 2, as has h6. Whether h7 is also associated with the tonoplast remains to be addressed. Are these isoforms unique to legumes is another interesting question. A rapid analysis by BLAST of databases of model plants belonging to legumes (Lotus japonicus, G. max, and Vigna unguiculata) or not (Arabidopsis, barley, tomato, N. benthamiana, and rice) revealed that except for L. japonicus and G. max, there is no close ortholog of Mth6 (or Mth7), suggesting that Mth6 and its orthologs may be specific to legumes.

In conclusion, three Trx h isoforms were found to be present in dry and germinating seeds of M. truncatula, h1, h2, and h6, differing by their biochemical properties, subcellular localization, and spatial distribution. Thus, they may serve different functions during germination. The abundant Mth1 and, to a lesser extent, Mth2, found in axes and cotyledons, may serve general functions of reserve mobilization, metabolism resumption, and ROS scavenging. Whether they only have redundant functions or also act on different protein targets remains to be addressed. Regarding Mth6, this isoform might be involved in the reduction of proteins stored in vacuoles upon imbibition, but it might also have specific membrane targets linked to its unusual location on the tonoplast of cotyledon cells. It would be of great interest to identify these targets.

MATERIALS AND METHODS

Materials

Surface-sterilized seeds of Medicago truncatula ‘Paraggio’ (Seedco Australia Co-Operative) were imbibed on filter paper (Whatman No. 1) in petri dishes (9 cm diameter) soaked with 3.5 mL of distilled water and allowed to germinate at 20°C in the dark during 48 h. Dry seeds or seeds imbibed for 14 h (before radicle protrusion) and 22 h (after radicle protrusion) were dissected in embryo axes and cotyledons. Alternatively, germinated seeds were transferred to soil and plants were allowed to grow at 20°C (16 h of light, 8 h of dark) with regular watering. Roots and leaves were harvested 21 d after the start of dry seed imbibition.

RNA Extraction, cDNA Cloning, and Overexpression of Recombinant Trxs h

Total RNAs were extracted from embryo axes and cotyledons from 14-h-imbibed seeds or roots and leaves from 21-d-old plants with the RNeasy plant kit (Qiagen) and further reverse transcribed using the Moloney murine leukemia virus reverse transcriptase (Promega) after a RQ1 DNase (Promega) treatment according to the manufacturer’s instructions. The coding regions corresponding to selected Trx h isoforms were amplified by PCR using cDNAs and the proofreading polymerase KOD HiFi DNA polymerase (Novagen), with primers (ORF sens and ORF anti) and at annealing temperature indicated in Supplemental Table S3, with a preliminary denaturation step of 4 min at 94°C, followed by a set of 35 cycles (94°C for 1 min, annealing temperature for 1 min, and 72°C for 1 min), and a final elongation step of 10 min at 72°C. The PCR products were resolved on 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). The vector is engineered to express target proteins as fusions to an N-terminal His tag that can be removed by enterokinase. Recombinant plasmids were introduced in Nova Blue Escherichia coli for multiplication and sequencing (MWG-Biotech) before transfer into BL21 (pLysS) cells for the production of proteins. Proteins were extracted in extraction buffer (50 mm phosphate, 5 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride, pH 7.5). For purification, recombinant Trxs h were trapped on either Q-Sepharose or S-Sepharose depending on their pI (Table II) and on Ni²⁺-chelating Sepharose (Amersham). Chromatography was carried out according to the manufacturer’s instructions. The concentration of each purified Trx was determined using its ε_M at 280 nm. For SDS-PAGE analysis, the His tag was removed using the enterokinase cleavage-capture kit (Novagen) following the manufacturer’s instructions.

Enzymatic Assays

Trx activity of recombinant protein was measured using the insulin or DTNB reduction assays as described previously (Holmgren, 1979; Jacquot et al., 1995) in the presence of DTT or NADPH and the recombinant NTRA originating from M. truncatula (Alkhalfioui et al., 2007a).

Protein Extraction, Gel Electrophoresis, and Western-Blot Analyses

Cotyledons and embryo axes from mature or germinating seeds as well as roots and leaves from 21-d-old plants were ground in 10 mm potassium acetate, pH 4.5, with 1 mm phenylmethylsulfonyl fluoride and 1 mm EDTA (10–20 mL g⁻¹ fresh weight). The resulting homogenate was centrifuged (40,000g for 30 min at 4°C). Protein contents of soluble fractions were determined using the Bradford reagent and bovine serum albumin as a standard (Bradford, 1976). Soluble proteins were then resolved on 1D or 2d gels. For 1D gels, 15 to 50 μg of proteins was loaded per lane on 15% (w/v) acrylamide gels by SDS-PAGE (Laemmli, 1970). For 2D gels, proteins were precipitated for 4 h at –20°C using 5 volumes of cold acetone or 20% TCA. The protein pellets were washed twice in 80% cold acetone. Proteins were thereafter resuspended in a rehydration solution for isoelectric focusing (6 m urea, 2 m thiourea, 4% CHAPS, 20 mm DTT, and 0.2% IPG buffer), and concentrations were checked using bovine serum albumin as a standard (Bradford, 1976). Proteins were loaded onto IPG strips (Bio-Rad) for isoelectric focusing: 150 to 200 μg was loaded on 7-cm strips (3–10 nonlinear pH gradient) for western-blot analysis or 350 to 400 μg was loaded on 17-cm strips (4–7 pH gradient) for the small proteome analysis. Isoelectric focusing was performed using the Protean IEF Cell (Bio-Rad) with the following parameters: 0 to 250 V linear gradient for 15 min; 250 to 4,000 V linear gradient for 4 h; and 4,000 V until 25,000 V/h was reached. The strips were then sequentially equilibrated at room temperature with 375 mm Tris-HCl, pH 8.8, 8 m urea, 20% glycerol, 130 mm DTT, and 2% SDS for 15 min and with the same buffer in which DTT was replaced by 135 mm iodoacetamide for another 20 min. Furthermore, the strips were placed on top of 15% acrylamide gels for SDS-PAGE.

After electrophoresis, proteins were either stained on the gels with colloidal Coomassie Brilliant Blue or transferred onto polyvinylidene fluoride membranes as described previously (Duval et al., 2002).

Membranes were probed with a 1:500 (v/v) dilution of a mix of antibodies raised against pea (Pisum sativum) Trx h3 and Trx h4 (Montrichard et al., 2003), and immunodetection was performed using the alkaline phosphatase assay in the presence of 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. For protein identification, spots of interest were excised manually and in-gel digested with trypsin. Peptides were analyzed by mass spectrometry (BiogenOuest; http://www.proteome.univ-rennes1.fr) or liquid chromatography-tandem mass spectrometry (INRA; http://www.angers-nantes.inra.fr/plates_formes_et_plateaux_techniques/plate_forme_bibs).

Determination of Subcellular Localization of Trx h6

Transient expression of Trx h6 in fusion to GFP or RFP in M. truncatula or Nicotiana benthamiana leaves was carried out using the Gateway cloning technology as described previously (Alkhalfioui et al., 2008). The full-length cDNA corresponding to Trx h6 was amplified by PCR from the recombinant pRSF2 plasmid with Att primers (Supplemental Table S3) 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 Trx h6 fused to GFP or RFP, the cDNAs were transferred using the Gateway LR Clonase enzyme mix into Destination binary vectors pK7FWG2 or pB7RWG2, respectively, allowing a constitutive transcription of the gene of interest under the control of a cauliflower mosaic virus 35S promoter (35S:Trxh6:GFP or 35S:Trxh6:RFP). These constructs were checked by restriction digest analysis. Recombinant plasmids 35S:Trx:GFP/RFP were then transferred into Agrobacterium tumefaciens (strain GV3101) by electroporation. 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 600 nm were used to infiltrate leaves of M. truncatula or N. benthamiana. For colocalization experiments, A. tumefaciens containing the recombinant binary plasmid 35S:Trxh6:RFP were infiltrated in leaves of M. truncatula together with A. tumefaciens containing a recombinant pGW5 binary plasmid, 35S:GFP:TIP1-1, allowing the expression of a GFP-fused TIP1-1, a marker protein of tonoplast (Boursiac et al., 2005). The latter plasmid was a gift of Dr. C. Maurel (INRA, CNRS, Université de Montpellier). Three to 4 d post agroinfiltration, cortical regions of leaf epidermal cells were observed by confocal microscopy using a Zeiss LSM510 microscope. Excitation/emission wavelengths were 488 nm/505 to 545 nm for GFP, 561 nm/575 to 615 nm for mRFP, and 561 nm/long-pass 650 nm for chloroplast autofluorescence. For some experiments, cells were plasmolyzed by incubation of the leaf samples in 0.45 m mannitol before observation.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DQ121442, DQ121443, FJ858140, FJ858141, FJ858142, FJ858143, FJ858144, and FJ858145 (Table II).

Supplemental Data

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

• Supplemental Figure S1. Alignment of Mth8 and Mth12 with their orthologs from Arabidopsis, Populus trichocarpa, and Triticum aestivum.

• Supplemental Figure S2. Kinetics of insulin reduction by recombinant Trxs h.

• Supplemental Figure S3. Kinetics of insulin reduction by Mth8 in the presence of DTT, GSH, or GSH plus Grx.

• Supplemental Figure S4. Western-blot detection of Mth6 and GFP in extracts of N. benthamiana leaves transiently overexpressing Mth6 fused to GFP.

• Supplemental Table S1. Prediction of subcellular localization of Trxs h of M. truncatula Jemalong.

• Supplemental Table S2. List of peptides identified in extracts of embryo axis and cotyledons from seeds of M. truncatula Paraggio.

• Supplemental Table S3. Primers used for the cloning of the coding regions of Trxs h in pRSF2 or pDON207 vectors.