A novel type of thioredoxin dedicated to symbiosis in legumes

Abstract

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.

Figure 1.

Darwin tree of M. truncatula and Arabidopsis Trx protein sequences. The protein sequences were deduced from nucleic sequences available for each species. The numbers of M. truncatula Trx sequences are indicated in Table I. The numbers of Arabidopsis Trx genes are (in parentheses): h1 (AT3G51030), h2 (AT5G39950), h3 (AT5G42980), h4 (AT1G19730), h5 (AT1G45145), h7 (AT1G59730), h8 (AT1G69880), h9 (AT3G08710), h10 (AT3G56420), CxxS1 (AT2G40790), CxxS2 (AT1G11530), f1 (AT3G02730), f2 (AT5G16400), m1 (AT1G03680), m2 (AT4G03520), m3 (AT2G15570), m4 (AT3G15360), o1 (AT2G35010), o2 (AT1G31020), x (AT1G50320), y1 (AT1G76760), and y2 (AT1G43560).

Figure 2.

Alignment of Trxs s with Trxs m and Trx h2 from M. truncatula. The protein sequences deduced from the coding regions of Trx s1 (DQ121444) and Trx s2 (DQ121445) were aligned with those of Trx h2 (DQ121443) and Trxs m. 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.

Figure 3.

Analyses of proteins by SDS-PAGE and western blot. For analyses of recombinant Trxs s, Trx s1r and Trx s2r were overexpressed in fusion with a His-tag in E. coli and purified. After the removal of the His-tag, proteins were resolved by 15% (w/v) SDS-PAGE and stained with Coomassie Blue (A) or transferred onto PVDF membranes (B). For SDS-PAGE analysis, 1 μg of each recombinant protein was loaded per lane. For western blotting, different quantities were loaded per lane ranging from 3 to 100 ng as indicated. For analyses of M. truncatula soluble proteins, they were extracted from whole seeds either dry (0 h) or imbibed for 14 h (before the radicle protrusion) or 22 h (after the radicle protrusion), leaves and roots from 20-d-old nonsymbiotic plants (−Sm), and leaves, roots, and functioning nodules from 20- to 25-d-old plants grown in symbiosis with S. meliloti (+Sm). They were resolved by 15% (w/v) SDS-PAGE and then transferred onto PVDF blots (B). Then 25 μg/lane was loaded for anti-s2 and anti-h blots and 50 μg/lane was loaded for anti-s1 blot. Blots were probed using the alkaline phosphatase assay with a 1:1,000 (v/v) dilution of antibodies raised against either a synthetic peptide derived from Trx s1 (Anti-s1) or the whole Trx s2r (Anti-s2). For anti-h blot, a mix of anti-h antibodies raised against P. sativum Trx h3 and Trx h4 was used at a 1:500 (v/v) dilution. Molecular masses of standard proteins are indicated in kilodaltons at the left. Data are representative of results obtained in three independent experiments.

Figure 4.

Disulfide reductase activity of Trxs determined by the DTNB reduction assay. Different concentrations of Trxs were incubated in the presence of 1.6 μm MtNTRA, 0.2 mm NADPH, and 100 μm DTNB in 30 mm Tris-Cl, pH 8. DTNB reduction was followed at 412 nm.

Figure 5.

Reduction of Trx h2r, Trx s1r, and Trx s2r by NTR plus NADPH or other reducing agents. Trxs were incubated in the absence (None) or the presence of DTT (2.5 mm for Trx h2r and Trx s1r or 20 mm for Trx s2r), 5 mm GSH, 0.2 mm NADPH plus 1 μm MtNTRA, or 0.2 mm NADPH alone, during 15 min at RT, after which any SH groups formed during this time were labeled with 5 mm mBBr. Proteins were finally resolved by 15% (w/v) SDS-PAGE and observed under UV light (black segments) before staining with Coomassie Blue (gray segments).

Figure 6.

Visualization of Trxs s:GFP in M. truncatula and N. benthamiana leaf epidermal cells. A, ER-like fluorescence pattern resulting from Trx s1:GFP expression in M. truncatula. B, ER-like fluorescence pattern resulting from Trx s2:GFP expression in N. benthamiana. C and D, Coexpression of Trx s1:GFP and Trx s2:GFP, respectively, with the ER marker RFP:HDEL. In cells coexpressing Trxs s:GFP and RFP:HDEL, near perfect colocalization is observed. Punctuate structures in the ER vicinity are indicated by arrowheads. E and F, Coexpression of Trx s1:GFP and Trx s2:GFP, respectively, with the Golgi marker Man1:RFP. Only very moderate colocalization between Trxs s:GFP and Man1:RFP is observed. C and E, M. truncatula leaf epidermal cells. D and F, N. benthamiana leaf epidermal cells. Scale bars = 5 μm.

Figure 7.

Trx s1 and Trx s2 gene expression. Quantitative RT-PCR experiments were conducted using total RNAs extracted from different organs: embryo axis and cotyledons of germinated seeds (imbibed for 22 h), leaves, and roots from 20-d-old nonsymbiotic plants (−Sm), and leaves, roots, and functioning nodules from 20- to 25-d-old plants grown in symbiosis with S. meliloti (+Sm). Amplification of the sequences of two constitutive genes (18S RNA and Msc27) was followed using Sybr Green. Amplification of Trx s genes was monitored with fluorescent Taqman probes. The results obtained were then expressed as 2^−ΔCT ± sd (ΔCT = CT of the gene of interest – mean of CTs of the constitutive genes). Data are representative of results obtained in three independent experiments.