Volume 7, Issue 1, Pages 30-44 (January 2014) NTR/NRX Define a New Thioredoxin System in the Nucleus of Arabidopsis thaliana Cells  Corinne Marchal, Valérie Delorme-Hinoux, Laetitia Bariat, Wafi Siala, Christophe Belin, Julio Saez-Vasquez, Christophe Riondet, Jean- Philippe Reichheld  Molecular Plant  Volume 7, Issue 1, Pages 30-44 (January 2014) DOI: 10.1093/mp/sst162 Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 1 Phylogenetic Tree of NRX. The accession numbers are as follows: AaNRX1, EGB06803; ApNRX, XP_001947195; AsNRX, ADY43508; AeNRX, EGI65199; AlNRX1, AFH64402; AlNRX2, EFH43567; AtNRX1, AEE33684; AtNRX2, AEE85880; BsNRX1, ACI16008; BtNRX, NP_001095606; CgNRX, EGV96743; CmNRX1, AAU04767; CrNRX1a, XP_001701995; CrNRX1b, XP_001701806; CrNRX2, XP_001701801; CrNRX3, EDO99555; EcNRX, XP_001502218; EsNRX1a, CBN76885; EsNRX1b, CBN79184; DrNRX, NP_001018431; HgNRX, EHB13353; HsNRX, NP_071908; MdNRX, XP_001364571; MmNRX, NP_032776; MnNRX1, XP_002504076; MtNRX1, ACJ85567; MtNRX2, XP_003603818; MtNRX3, XP_003603563; NveNRX, XP_001634149; NviNRX, XP_001607483; QsNRX1, AAS02080; OsNRX1a, NP_001050329; OsNRX1b, AAU89249; OsNRX1c, EEC75442; OsNRX1d, NP_001050331; OsNRX2, NP_001044503; OsNRX3, EEC77960; PhtNRX1, XP_002183102; PsNRX1, ABK25413; PsNRX2-3, ABK25089; PtNRX1a, XP_002314537; PtNRX1b, XP_002314534; PtNRX1c, XP_002314533; PtNRX1d, EEF00707; PtNRX1e, XP_002314535; PtNRX2, XP_002306954; PtNRX3, XP_002330779; RcNRX1, XP_002525368; RcNRX2, XP_002510593; RtNRX, NP_001101755; SbNRX1, XP_002467709; SbNRX2, XP_002467708; SbNRX3, XP_002448495; SmNRX1, EFJ30324; SsNRX, NP_001167329; TgNRX1a, EEE25737; TgNRX1b, EEE24650; TsNRX1, EFV57630; VcNRX1, EFJ40226; VvNRX1a, XP_002263480; VvNRX1b, CBI28536; VvNRX1c, XP_002262828; VvNRX1d, XP_002262857; VvNRX1e, XP_002264954; VvNRX2, XP_002285895; VvNRX3, CBI20806; XlNRX, NP_001086161; ZmNRX1a, NP_001105407; ZmNRX1b, NP_001130856; ZmNRX2, NP_001131397. Aa, Aureococcus anophagefferens; Ap, Acyrthosiphon pisum; As, Ascaris suum; Ae, Acromyrmex echinatior; Al, Arabidopisis lyrata; At, Arabidopsis thaliana; Bs, Bodo saltans; Bt, Bos taurus; Cg, Cricetulus griseus; Cm, Cucumis melo; Cr, Chlamydomonas reinhardtii; Dr, Danio rerio; Ec, Equus caballus; Es, Ectocarpus siliculosus; Hg, Heterocephalus glaber; Hs, Homo sapiens; Md, Monodelphis domestica; Mm, Mus musculus; Mn, Micromonas sp. RCC299; Mt, Medicago truncatula; Nve, Nematostella vectensis; Nvi, Nasonia vitripennis; Os, Oryza sativa; Pht, Phaeodactylum tricornutum CCAP 1055/1; Ps, Picea sitchensis; Pt, Populus trichocarpa; Qs, Quersus suber; Rc, Ricinus communis; Rn, Rattus norvegicus; Sb, Sorghum bicolor; Sm, Selaginella moellendorffii; Ss, Salmo salar; Tg, Toxoplasma gondii; Ts, Trichinella spiralis; Vc, Volvox carteri f. nagariensis; Vv, Vitis vinifera; Xl, Xenopus laevis; Zm, Zea mais. Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 2 Comparative Domain Organization of Different Types of NRX. TRX domains are represented by shaded gray boxes and undefined domains as white boxes. CTD, Cysteine-rich C-terminal domain; PDI b’, PDI-like C-terminal domain. Canonical TRX active sites are represented as black bars. Atypical TRX-like active sites are shown as gray bars. Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 3 Activity of NRX Determined by the Insulin–Disulfide Reduction Assay. (A) Purification of the recombinant (His)6-NRX. 1 μg of NRX1, NRX1-ΔCterm, and NRX2 was loaded onto an SDS–PAGE gel. M, molecular weight marker. (B) Insulin–disulfide reduction of the recombinant NRX1 (circles), NRX1-ΔCterm (crosses), NRX2 (triangles), and TRXh3 (squares) was measured at pH 7.0 and 20°C. 1 μM of each enzyme was used in the presence of 1mM DTT and 0.13mM insulin. The baseline of insulin reduction by DTT was used as a control (black line). The time course of insulin precipitation was measured at 650nm. Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 4 Reduction of NRX1 by NTRA. (A) Reduction activity of NRX by NTR determined by the insulin–disulfide reduction assay. The activity was assayed with 10 μM FAD, 0.5mM NADPH, different concentrations of NRX1 proteins, and 1 μM of AtNTRA. The consumption of NADPH was followed spectrophotometrically at 340nm at 20°C during 15min and the initial rate of consumption was used to measure the activity. (B) Km determination NTRA for NRX1 at pH 7.0 and 20°C. Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 5 Complementation of the Budding Yeast trx1trx2 Mutant by NRX. AtNRX1 is able to partially complement the H2O2 tolerance of the trx1trx2 mutant. Cells expressing AtNRX1, AtNRX2, yTRX1, and yTRX2 from the pGK1 promoter of the pFL61 high-copy number expression vector were grown to a density of 107 cells per ml and plated on YNB agar medium at the indicated OD600nm in the presence or absence of methionine (–Met, +Met) or in the presence of methionine sulfoxide (+MetO) as sole source of sulfur. Cells were also plated on YNB agar medium containing H2O2 at various concentrations. Plates were incubated for 4 d at 28°C. Several independent transformed yeast clones were tested with very similar complementation profiles. Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 6 Cytosolic and Nuclear Subcellular Localization of NRX and NTR. (A–C) Whole mount root tip immunolocalization of NRX1. (A) DAPI staining in blue; (B) anti-NRX1 Ab in yellow; (C) merge. (D–H) Squashed root tip immunolocalization of NRX1. (D) DAPI staining in blue; (E) anti-NRX1 Ab in yellow; (F) merge; the red arrow indicates where the intensities were measured for both channels DAPI and Alexa (cf graph in (I)). (G, H) are controls without the anti-NRX1 primary antibody; (G) DAPI staining in blue; (H) Alexa secondary antibody only in yellow. (I) This graph gives the intensity according to the distance (μm) along the arrow displayed in (F) for both DAPI and Alexa channels. NRX1 is present in the cytoplasm and the nucleus but excluded from the nucleolus. (J–L) crop images of a whole mount root tip immunolocalization of NTR. (J) DAPI staining in blue; (K) anti-NTR Ab in yellow; (L) merge. Contrary to NRX1, NTR is more abundant in the cytoplasm than in the nucleus. NTR is also excluded from the nucleolus. (M) Detection of NRX1 and NTR proteins in subcellular cytosolic (C) and nuclear (N) extracts of Arabidopsis flower buds. Immunodetections of cytosolic thioredoxins TRXh3 and TRXh5, and nuclear nucleolin Nuc1 were used to evaluate the purity of the subcellular fractions. Bar is 2 μm for (A–F) and 7 μm for (G, H, J–L). Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 7 Dimerization of the AtNRX1 Protein. (A) Gel filtration chromatographic analysis of crude protein extracts from flower buds separated under 0.5M KCl buffer conditions. AtNRX1 protein was detected by Western blot. Numbered lines correspond to the protein fractions (from 49 to 79). The peak position of alcohol dehydrogenase (ADH, 158kDa) and bovine serum albumin (BSA, 67kDa) markers are indicated by black arrows. The white arrow at the fraction 65 indicates the estimated size of the AtNRX1 protein peak. Crude, crude protein extract (5 μl) before gel filtration. (B) Gel filtration chromatographic analysis of the recombinant AtNRX1 protein. The peak position of coalbumine (75kDa) and β-amylase (200kDa) markers are indicated by dashed lines. 5 μl of each of fractions 20 to 34 was analyzed under reducing conditions by Western blot using the anti-NRX1 antibody. Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

Figure 8 Affected Pollen Fertility in nrx1 Mutants. (A) Segregation analysis of nrx1-401, nrx1-340, and nrx2-735 mutant alleles. % progeny, percentage of plants in the progeny having the given nrx1 mutant genotype (Wt, wild-type; Ht, heterozygous; Ho, homozygous). n, number of progeny analyzed by PCR. P-values<0.005 (*) and <0.001 (**) were considered to be statistically significant. NS, P-values>0.05 were considered to be non-significant. (B) Reciprocal cross between NRX1/nrx1-401 and Col-0. Kan, number of plants being resistant (R) or sensitive (S) to kanamycine. %, percent of R or S plants; n, number of plants analyzed; P-values<0.005 (*) were considered to be statistically significant. NS, P-values>0.05 were considered to be non-significant. (C) NRX expression in pollen. Semi-quantitative RT–PCR was performed on RNA extracted from mature pollen and mature flowers for 30, 35, and 40 cycles. SPIK was used as a pollen-specific control gene. Note that a high level of SPIK cDNA is also present in mature flowers. EF1α is used as a constitutive marker gene. Molecular Plant 2014 7, 30-44DOI: (10.1093/mp/sst162) Copyright © 2013 The Authors. All rights reserved. Terms and Conditions