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• W1991444355 abstract "Thioredoxins type h are classified into three subgroups. The subgroup II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollenpistil interaction processes or particularly in S-RNase-based self-incompatibility. Thioredoxins type h are classified into three subgroups. The subgroup II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollenpistil interaction processes or particularly in S-RNase-based self-incompatibility. Thioredoxins (Trxs) 2The abbreviations used are: Trx, thioredoxin; Trx h, thioredoxin type h; NTR, NADPH-dependent thioredoxin reductase; ECM, extracellular matrix; TT, transmitting tract; GFP, green fluorescent protein; mBBr, monobromobimane; PAO, 4-aminophenylarsine oxide; SI, self-incompatibility/incompatible; SC, self-compatible; GST, glutathione S-transferase; NaTrxhrec, recombinant NaTrxh; NaTTS, N. alata transmitting tissue-specific protein; DTT, dithiothreitol; BB, binding buffer; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid. are small conserved proteins that play an important role in cellular redox regulation. When the Trx active site WCGPC is reduced, it is able to reduce the disulfide bonds of target proteins. Trxs are widely distributed in nature from prokaryotes to eukaryotes. In photosynthetic organisms, Trxs have been shown to be highly polymorphic and to participate in several central cellular processes (1Arnér E.S.J. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2001) Google Scholar, 2Marchand C. Le Maréchal P. Meyer Y. Miginicac-Maslow M. Issakidis-Bourguet E. Decottignies P. Proteomics. 2004; 4: 2696-2706Crossref PubMed Scopus (169) Google Scholar). The diversity of physiological roles in which Trxs participate depends entirely on the target proteins (3Holmgren A. Ann. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 4Eklund H. Gleason F.K. Holmgren A. Proteins. 1991; 11: 13-28Crossref PubMed Scopus (328) Google Scholar, 5Meyer Y. Verdoucq L. Vignols F. Trends Plant Sci. 1999; 4: 388-394Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The 20 genes encoding Trxs in the Arabidopsis thaliana genome (6Meyer Y. Vignols F. Reichheld J.-P. Methods Enzymol. 2002; 347: 394-402Crossref PubMed Scopus (119) Google Scholar) and the proteomic analysis (2Marchand C. Le Maréchal P. Meyer Y. Miginicac-Maslow M. Issakidis-Bourguet E. Decottignies P. Proteomics. 2004; 4: 2696-2706Crossref PubMed Scopus (169) Google Scholar) of their targeted proteins by Trx h3 in A. thaliana reinforce the wide range of functions in which these proteins are involved. In plants, five different types of Trxs have been described: m, f, x, o, and h. Types m, f, and x are chloroplastic, whereas type o Trxs are mitochondrial and type h Trxs (Trx h) are assumed to be localized in the cytoplasm (5Meyer Y. Verdoucq L. Vignols F. Trends Plant Sci. 1999; 4: 388-394Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 7Johnson T.C. Cao R.Q. Kung J.E. Buchanan B.B. Planta. 1987; 171: 321-331Crossref Scopus (47) Google Scholar, 8Hartman H. Syvanen M. Buchanan B.B. Mol. Biol. Evol. 1990; 7: 247-254PubMed Google Scholar, 9Hodges M. Miginiac-Maslow M. Decottignies P. Jacquot J.-P. Stein M. Lepeniec L. Cretin C. Gadal P. Plant Mol. Biol. 1994; 26: 225-234Crossref PubMed Scopus (46) Google Scholar, 10Rivera-Madrid R. Mestres D. Marinho P. Jacquot J.-P. Decottignies P. Muginiac-Maslow M. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5620-5624Crossref PubMed Scopus (113) Google Scholar, 11Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (223) Google Scholar). The mitochondrial and cytoplasmic Trxs are reduced by mitochondrial (11Laloi C. Rayapuram N. Chartier Y. Grienenberger J.M. Bonnard G. Meyer Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14144-14149Crossref PubMed Scopus (223) Google Scholar) or cytoplasmic (12Florencio F.J. Yee B.C. Johnoson T.C. Buchanan B.B. Arch. Biochem. Biophys. 1988; 266: 496-507Crossref PubMed Scopus (119) Google Scholar) NADPH-dependent thioredoxin reductases (NTR). Although Trxs h have traditionally been considered to be cytoplasmic proteins, recent analyses (13Gelhaye E. Rouhier N. Jacquot J.-P. Plant Physiol. Biochem. 2004; 42: 265-271Crossref PubMed Scopus (122) Google Scholar) revealed that they can be further differentiated into three subgroups. Subgroup II includes Trxs h with N-terminal extensions. The biochemical function of these extensions remains unclear, and the available algorithms do not predict any targeting signal. Gelhaye et al. (14Gelhaye E. Rouhier N. Gérard J. Jolivet Y. Gualberto J. Navrot N. Ohlsson P.I. Wingsle G. Hirasawa M. Knaff D.B. Wang H. Dizengremel P. Meyer Y. Jacquot J.-P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14545-14550Crossref PubMed Scopus (204) Google Scholar) reported on a Trx h subgroup II from poplar (Populus tremula), PtTrxh2, the N-terminal extension of which is necessary to target this Trx h to mitochondria. In addition, RPP13-1, a Trx h present in the rice (Oryza sativa) phloem sap, mediates its own transport from cell to cell through plasmodesmata only if its N-terminal extension is present (15Ishiwatari Y. Honda C. Kawashima I. Nakamura S.I. Hirano H. Mori S. Fujiwara T. Hayashi H. Chino M. Planta. 1995; 195: 456-463Crossref PubMed Scopus (160) Google Scholar, 16Ishiwatari Y. Fujiwara T. McFarland K.C. Nemoto K. Hayashi H. Chino M. Lucas W.J. Planta. 1998; 205: 12-22Crossref PubMed Scopus (152) Google Scholar). Here, we report an analysis of a Trx h that belongs to the subgroup II, from Nicotiana alata, called NaTrxh. The N-terminal extension of NaTrxh was not recognizable as a secretion signal, and yet we demonstrated that NaTrxh accumulates in the extracellular matrix (ECM) of the stylar transmitting tract (TT) in N. alata. Moreover, fusion of NaTrxh to the green fluorescent protein (GFP) directed secretion of this fusion protein in two heterologous systems. We used two separate biochemical approaches, monobromobimane (mBBr) labeling and 4-aminophenylarsine oxide (PAO) chromatography, to test potential substrates of NaTrxh. S-RNase was clearly among the proteins reduced by NaTrxh, raising the possibility that it is involved in pollination, particularly in self-incompatibility (SI). Plant Materials—Nicotiana plumbaginifolia (inventory number TW107, accession number 43B) was obtained from Tobacco Germoplasm Collection number TW107, Crops Research Laboratory, U. S. Department of Agriculture-Agricultural Research Service, Oxford, NC. Self-compatible (SC) N. alata cv Breakthrough was obtained from Thompson and Morgan, Jackson, NJ. Self-incompatible (SI) N. alata S105S105 and SC10SC10 have been described previously (17Murfett J. Bourque J.E. McClure B.A. Plant Mol. Biol. 1995; 29: 201-212Crossref PubMed Scopus (17) Google Scholar, 18Murfett J. Strabala T.J. Zurek D.M. Mou B. Beecher B. McClure B.A. Plant Cell. 1996; 8: 943-958Crossref PubMed Scopus (131) Google Scholar, 19Zurek D.M. Mou B. Beecher B. McClure B.A. Plant J. 1997; 11: 797-808Crossref PubMed Scopus (59) Google Scholar). Nicotiana benthamiana and A. thaliana Columbia (Col-0) ecotype plants were grown under greenhouse conditions using a 16:8 photoperiod at 22 °C. Recombinant NaTrxh Overexpression and Purification—The NaTrxh cDNA with BamHI and EcoRI sites was generated using the following primers: forward, 5′-CGCGCGGATCCATGGGATCGTATCTTTCAA-3′; reverse, 5′-GCGCGCGGGAATTCAATTTATTGGACATGAAA-3′. The PCR product was cloned in-frame with the glutathione S-transferase (GST) into the pGEX 4T-2 expression vector (Amersham Biosciences). The GST:NaTrxh fusion protein was separated by batch affinity chromatography using glutathione-Sepharose 4B (Amersham Biosciences). NaTrxh was released from GST by thrombin digestion (Amersham Biosciences). NaTrxh-GFP Fusion Constructs and Transient Expression—An NaTrxh cDNA with BamHI and NcoI sites was produced by PCR and fused in-frame to the N-terminal of the GFP in the pHBT vector (20Sheen J. Hwang S. Niwa Y. Kobayashi H. Galbraith D.W. Plant J. 1995; 8: 777-784Crossref PubMed Scopus (328) Google Scholar). Primers used are as follows: forward, 5′-CGCGCGGATCCATGGGATCGTATCTTTCAA-3′; reverse, 5′-AGCCATGGTTGGACATG-3′. The NaTrxh-GFP fusion was cloned into pBIN19 (21Jefferson R.A. Genet. Eng. 1988; 10: 247-263Google Scholar) under control of the 35S cauliflower mosaic virus (CaMV35S) promoter. After Agrobacterium tumefaciens pGV2260 (22Deblaere R. Bytebier B. De Greve H. Deboeck F. Schell J. Van Montagu M. Leemans J. Nucleic Acids Res. 1985; 13: 4777-4788Crossref PubMed Scopus (597) Google Scholar) transformation, an agroinfiltration of N. benthamiana leaves was carried out, as described previously (23Schöb H. Kunz C. Meins Jr., F. Mol. Gen. Genet. 1997; 256: 581-585Crossref PubMed Scopus (122) Google Scholar, 24Llave C. Kasschau K.D. Carrington J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13401-13406Crossref PubMed Scopus (300) Google Scholar). The protein expression was observed by confocal microscopy after 3 days. A. thaliana Col-0 transformation was carried out by bombarding (25Sanford J.C. Physiol. Plant. 1990; 79: 206-209Crossref Scopus (149) Google Scholar) 2-week-old plantlets (grown aseptically in 0.5× Murashige and Skoog with 1% sucrose) with DNA-coated tungsten particles (Tungsten M-17, Bio-Rad) containing the NaTrxh-GFP construct cloned into the pHBT vector (20Sheen J. Hwang S. Niwa Y. Kobayashi H. Galbraith D.W. Plant J. 1995; 8: 777-784Crossref PubMed Scopus (328) Google Scholar). The protein expression was observed by confocal microscopy after 1 week. N. alata Total Protein Extracts—Total protein extracts from mature styles including stigmas, anthers, ovaries, sepals, petals, and young leaves from N. alata were obtained using 0.05 m sodium acetate, pH 5.0, 0.05 m NaCl, 1% (v/v) 2-mercaptoethanol as extraction buffer. Sequential Style Protein Extractions—N. alata styles were treated as described previously (26Wu H.M. Wong E. Ogdahl J. Chueung A.Y. Plant J. 2000; 22: 165-176Crossref PubMed Scopus (137) Google Scholar), with the following modifications. Hand-bisected N. alata styles (styles + stigmas) were submerged sequentially in two different salt concentration buffers for 2.5 h at 4 °C: (a) low salt buffer (100 mm Tris·HCl, pH 8.0, 2 mm Na2S2O4); (b) high salt buffer (400 mm NaCl, 40 mm Tris·HCl, pH 8.0, 2 mm Na2S2O4). Finally, the styles were ground under liquid N2 and homogenized in 400 mm NaCl, 40 mm Tris·HCl, pH 8.0, 2 mm Na2S2O4, 1% Triton X-100. Protein Assay—Protein concentrations were determined as described previously (27Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar), using bovine serum albumin (BSA) as standard. Reductase Activity Assay—Recombinant NaTrxh (NaTrxhrec) and recombinant Escherichia coli thioredoxin (Sigma) were evaluated for their ability to reduce insulin disulfide bonds as described previously (28Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar). The ability of the NADPH/NTR system to reduce NaTrxhrec was evaluated as follows: NaTrxhrec (2.5 μg) and E. coli Trx (2.5 μg) in 50 mm Tris·HCl, pH 7.9, were incubated at 37 °C with 2.0 μg of recombinant E. coli NTR and 0.125 μmol of NADPH in a final volume of 100 μl. After incubation, 0.2 μmol of mBBr in 10 μl of acetonitrile was added, and the samples were incubated for 20 min at room temperature. Proteins were boiled for 5 min in SDS sample buffer free of reducing agents, separated in 10-20% gradient SDS-PAGE, and visualized under UV light (Fluor-S, Fuji Corp Saddle Brook, NJ). NaTrxh-Affi-Gel Affinity Column and Purification of Anti-NaTrxh Antibody—NaTrxhrec (16 mg) was immobilized to Affi-Gel-10 (Bio-Rad), as recommended by the manufacturer. The anti-NaTrxhrec serum was precipitated with 50% saturated ammonium sulfate. After desalting, this IgG fraction was passed over the NaTrxh-Affi-Gel affinity column. Specific antibodies against NaTrxhrec were eluted with 50 mm glycine, 50 mm NaCl, pH 2.6. The samples were neutralized by the addition of 1 m Tris. BSA-Affi-Gel and Gly-Affi-Gel Columns—BSA (20 mg) in 0.1 m MOPS, pH 7.5, was immobilized to Affi-Gel-15 (Bio-Rad), as recommended by the manufacturer. Gly-Affi-Gel column was made by blocking Affi-Gel-15 with 1.0 m glycine, pH 8.0, as recommended by the manufacturer. Phylogenetic Analysis—Amino acid sequences of plant Trxs were aligned by Clustal X (29Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35497) Google Scholar). Based on the alignment, a phylogenetic tree was constructed by the neighbor-joining method (30Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar). A phylogenetic test was done based on 1,000 bootstrap replicates. Phylogenetic analysis was conducted using MEGA version 2.1 (31Kumar S. Tamura K. Jakobsen I.B. Nei M. Bioinformatics. 2001; 17: 1244-1245Crossref PubMed Scopus (4553) Google Scholar). The GenBank™ accession numbers used for this analysis are as follows: Trx type f, A. thaliana (Q9XFH8), Pisum sativum (X63537), and Spinacia oleracea (X14959); Trx-like proteins, Hordeum bulbosum (AF159385), Lolium perenne (AF159387), Phalaris coerulescens (AF159388), Secale cereale (AF159386), A. thaliana (AAG51342); Trxs type h, Ipomea batatas (AY344228), A. thaliana (AAG52561, AAD39316, S58119, S58118, S58123, S58120, and P29448), Brassica napus (U59379), O. sativa (D26547), N. alata (NaTrxh) (DQ021448), Nicotiana tabacum (Q07090 and X58527); Trxs type m, A. thaliana (AAF15949, O48737, Q9SEU6, and AAF15950), P. sativum (X76269), S. oleracea (X51462), Zea mays (L40957), Trx type x, A. thaliana (AAF15952); and Trx type o, A. thaliana (AF396650). Protein Gel Blot Analysis and Immunostaining—Proteins were fractionated in a 12.5% SDS-PAGE, blotted onto nitrocellulose, and immunostained with anti-S105-RNase (1:10,000 dilution), anti-NaTTS (1:10,000 dilution) (32Cruz-García F. Hancock C.N. Kim D. McClure B. Plant J. 2005; 42: 295-304Crossref PubMed Scopus (64) Google Scholar), or anti-NaTrxhrec (1:1,000 dilution). In Vitro Identification of NaTrxh Target Proteins—Reduction of the disulfide bonds of target proteins was determined using the two-dimensional SDS-PAGE system (33Yano H. Wong J.H. Lee Y.M. Cho M.J. Buchanan B.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4794-4799Crossref PubMed Scopus (205) Google Scholar) using the low salt buffer extract without Na2S2O4. Before the first dimension, proteins are labeled with mBBr after non-reducing (control) or reducing (with dithiothreitol (DTT) or NaTrxhrec plus NADPH and NTR) conditions. The second dimension gel was observed under UV light (Fluor-S, Fuji Corp., Saddle Brook, NJ). Low salt protein extracts from N. alata S105S105 in 50 mm Tris·HCl, pH 7.9, were passed over PAO resin (Invitrogen) after reduction by NaTrxhrec, 20 mm 2-mercaptoethanol, or without reduction. After washing, the unbound fraction was collected. Bound proteins were eluted by sequentially washing the resin twice with 1 mm 2-mercaptoethanol containing buffer, five times with 5 mm 2-mercaptoethanol, and three times with 500 mm 2-mercaptoethanol buffer. All the fractions were concentrated by lyophilization and were analyzed by 12.5% SDS-PAGE. Gels were blotted onto nitrocellulose and immunostained with anti-S105-RNase antibody. In Vitro Protein-Protein Interactions by Affinity Chromatography— Protein extracts (1 mg) from N. alata S105S105 styles were obtained with binding buffer (BB, 50 mm Tris·HCl, pH 7.9) and passed over the NaTrxh-Affi-Gel affinity column. After recovering the unbound fraction, 10 bed volume washes were done with BB, and then the column was sequentially washed as follows: (a) BB plus 1% Tween 20, (b) BB plus 0.1 m NaCl, and (c) BB plus 0.2 m NaCl. Stronger interacting proteins were eluted with elution buffer (50 mm glycine, 50 mm NaCl, pH 2.6). The samples were neutralized by the addition of 1 m Tris. Fractions were concentrated by cold acetone precipitation and were analyzed by 12.5% SDS-PAGE. Gels were blotted onto nitrocellulose and immunostained with anti-S105-RNase antibody. Microscopy and Immunolabeling—N. alata SC10SC10 styles were harvested and fixed in 4% (v/v) formaldehyde in phosphate-buffered saline, dehydrated in an ethanol series, and embedded in Paraplast Plus (Poly-sciences Inc., Warrington, PA). Sections of 6-7 μm were blocked with phosphate-buffered saline plus 3% BSA, 0.01% sodium azide, 0.1% Triton X-100 for 4 h at 4°C. Sections were simultaneously incubated with the primary rabbit anti-NaTrxhrec antibody (1:50 dilution) and the primary mouse anti-SC10-RNase antibody (1:1,000 dilution) at 4 °C overnight. Sections were then incubated with both secondary goat anti-rabbit Alexa Fluor 568-fluorochrome (magenta signal) conjugated and goat anti-mouse fluorescein isothiocyanate-fluorochrome (green signal) conjugated antibodies for 4 h at 4°C. Sections were observed using confocal fluorescence microscopy. Isolation of the Trx h cDNA—We isolated a cDNA (AFLP25F), which is differentially expressed between N. alata cv Breakthrough, an SC mutant plant that does not express the S-RNase, and SC N. plumbaginifolia. The full-length cDNA sequence of this transcript (Fig. 1A) showed extensive sequence similarity with Trx genes from plants. The predicted open reading frame contains the sequence WCGPC, described as the conserved Trx active site (3Holmgren A. Ann. Rev. Biochem. 1985; 54: 237-271Crossref PubMed Google Scholar, 4Eklund H. Gleason F.K. Holmgren A. Proteins. 1991; 11: 13-28Crossref PubMed Scopus (328) Google Scholar). To identify the type of Trx encoded by the AFLP25F cDNA (i.e. m, f, x, or h), we performed a phylogenetic analysis using the deduced AFLP25F amino acid sequence and other plant Trxs. The phylogenetic tree in Fig. 1B displays five major groups. Four of those groups correspond to organellar thioredoxins (f, m, x, and o types). The AFLP25F protein sequence clearly clusters with Trxs h, subgroup II. The AFLP25F product was named NaTrxh. To evaluate the disulfide reductase activity of NaTrxh, we expressed this gene in E. coli as a GST fusion protein. Fig. 2A shows the recombinant NaTrxh (NaTrxhrec) purified after cleavage from GST. Its ability to reduce insulin disulfide bonds using DTT as an electron donor (28Holmgren A. J. Biol. Chem. 1979; 254: 9627-9632Abstract Full Text PDF PubMed Google Scholar) is shown in Fig. 2B. The results show that NaTrxhrec was able to reduce insulin disulfide bridges with a similar qualitative activity profile as the recombinant E. coli Trx (34Hoog J.O. von Bahr-Lindstrom H. Josephson S. Wallace B.J. Kushner S.R. Jornvall H. Holmgren A. Biosci. Rep. 1984; 4: 917-923Crossref PubMed Scopus (30) Google Scholar). Furthermore, when NaTrxhrec was incubated with NADPH and recombinant E. coli NTR, the thiol groups of the reduced NaTrxhrec were labeled with mBBr. Fig. 2C shows NaTrxhrec and recombinant E. coli Trx sulfhydryl labeling in a time-dependent manner only when NADPH and NTR were present. Thus, similar to other Trxs h, oxidized NaTrxhrec can be regenerated by the addition of NADPH and NTR (12Florencio F.J. Yee B.C. Johnoson T.C. Buchanan B.B. Arch. Biochem. Biophys. 1988; 266: 496-507Crossref PubMed Scopus (119) Google Scholar, 35Baumann U. Juttner J. CMLS Cell. Mol. Life Sci. 2002; 59: 1042-1057Crossref PubMed Scopus (41) Google Scholar). NaTrxh Expression Pattern in N. alata—To analyze the expression pattern of NaTrxh in plants, we prepared specific affinity-purified anti-NaTrxh antibodies. Fig. 3A (right panel) shows that this antibody detects specifically NaTrxhrec with no cross reaction to E. coli Trx. This antibody was used to analyze the presence of the NaTrxh in different tissues of N. alata. NaTrxh protein is detected in all the tissues analyzed (Fig. 3B, right panel). This protein is particularly abundant in floral tissues including petals, ovaries, and styles, and it is present in lower levels in anthers, sepals, and leaves. Biochemical fractionation studies suggest an extracellular location for NaTrxh. SI N. alata S105S105 styles were hand-bisected, and the secreted proteins that accumulate in this tissue were differentially extracted using sequential washes with low salt and high salt buffers, as described (26Wu H.M. Wong E. Ogdahl J. Chueung A.Y. Plant J. 2000; 22: 165-176Crossref PubMed Scopus (137) Google Scholar). Using this procedure, soluble ECM proteins are eluted by a low salt buffer, whereas more tightly bound proteins are released only after the high salt buffer wash. The extraction profile obtained for the NaTrxh protein was similar to S105-RNase and to the transmitting tissue-specific protein of N. alata (NaTTS) (Fig. 4). It is known that these two proteins are secreted and that they accumulate in the stylar TT ECM. NaTrxh is eluted with the higher molecular mass (i.e. 55-110-kDa) NaTTS isoforms (Fig. 4C, LS lane) that are thought to be present in the ECM (26Wu H.M. Wong E. Ogdahl J. Chueung A.Y. Plant J. 2000; 22: 165-176Crossref PubMed Scopus (137) Google Scholar), suggesting that NaTrxh is also secreted into the ECM of the stylar TT. NaTrxh Is Secreted into the Extracellular Matrix of the Transmitting Tissue—To further investigate NaTrxh localization and its secretion to the stylar TT ECM, we performed an immunohistochemical analysis using three-dimensional confocal microscopy. Cross sections of SI N. alata SC10SC10 styles were simultaneously immunolabeled with a mouse anti-SC10-RNase antibody (32Cruz-García F. Hancock C.N. Kim D. McClure B. Plant J. 2005; 42: 295-304Crossref PubMed Scopus (64) Google Scholar) and with the affinity-purified polyclonal anti-NaTrxh antibody. Fig. 5, A2, A4 and A6, show that both NaTrxh and SC10-RNase colocalized outside of the TT cells, indicated by the yellow signal generated from the mixture of both green (SC10-RNase) and magenta (NaTrxh) fluorochrome signals. To show that the anti-NaTrxh antibody is specifically reacting with NaTrxh in the TT cells, we pretreated the antibody prior to apply to the tissue with NaTrxhrec. As shown in Fig. 5, A3 and A5, after pretreatment, only the green signal of SC10-RNase was detected. The experiments shown in Figs. 4 and 5A demonstrate that NaTrxh is secreted into the ECM. However, NaTrxh does not possess a canonical secretory signal peptide, and different secretion signal algorithms give conflicting results. For example, the Bendtsen neural network algorithm (36Bendtsen J.D. Nielsen H. von Heijne G. Brunak S. J. Mol. Biol. 2004; 340: 783-790Crossref PubMed Scopus (5643) Google Scholar) does not predict any signal peptide, whereas the hidden Markov model algorithm predicts a signal peptide with a probability of 0.953 with a maximal cleavage site probability of 0.593 between amino acid residues 16 and 17 (Fig. 1A). Likewise, the Secretome 1.0 predictor (37Bendtsen J.D. Jensen L.J. Blom N. von Heijne G. Brunak S. Protein Eng. Des. Sel. 2004; 17: 349-356Crossref PubMed Scopus (944) Google Scholar) predicts that NaTrxh (NN-score 0.874) is a non-classical secreted protein. NaTrxh Sequence Contains the Information for Its Secretion—To further corroborate that NaTrxh is indeed secreted in plants, an NaTrxh-GFP fusion protein expressed from the CaMV35S promoter (20Sheen J. Hwang S. Niwa Y. Kobayashi H. Galbraith D.W. Plant J. 1995; 8: 777-784Crossref PubMed Scopus (328) Google Scholar) was constructed. This construct was used to analyze GFP expression pattern in transient assays in N. benthamiana and A. thaliana leaves (Fig. 5B). When the NaTrxh-GFP fusion protein is expressed, most of the GFP protein accumulates in the cell wall in either N. benthamiana (Fig. 5, B2 and B4) or A. thaliana (Fig. 5, B10 and B12). As expected, the GFP protein without fusion, used as control, was mainly localized in the cytoplasm (Fig. 5, B6 and B8). Thus, NaTrxh is sufficient to cause secretion of GFP, supporting the idea that this protein is targeted outside of the cell in N. alata. S-RNase Is Reduced by NaTrxh in Vitro—To investigate possible substrates for NaTrxh, we performed in vitro reduction reactions with stylar proteins and NaTrxhrec plus NADPH and NTR. Reduced sulfhydryls were labeled with the fluorescent probe mBBr and visualized after separation in a two-dimensional SDS-PAGE system in which non-reducing conditions were used for the first dimension and reducing ones were used for the second dimension (33Yano H. Wong J.H. Lee Y.M. Cho M.J. Buchanan B.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4794-4799Crossref PubMed Scopus (205) Google Scholar). For these experiments, we used the low salt extractable ECM stylar protein fraction from SI N. alata S105S105 prepared without a reducing agent in the extraction buffer. As shown in Fig. 6, A and B, there is a small amount of mBBr labeling where S105-RNase is the most prominent labeled protein observed (Fig. 6, B and G). Proteins reduced after the first dimension show altered mobility and shift off the diagonal in the second dimension. If reduction is incomplete, two spots are visible, as seen for S105-RNase (Fig. 6, A and B). Proteins fully reduced with DTT prior to mBBr labeling show brighter fluorescence and appear as a single spot after two-dimensional SDS-PAGE (Fig. 6, C and D). As expected, many proteins are reduced by DTT, and mBBr labeling is visible along the entire diagonal. Treatment with purified NaTrxhrec, NADPH, and NTR caused a specific increase in mBBr labeling of S105-RNase similar to the one obtained by DTT (Fig. 6, E and F). S105-RNase changed its mobility after reduction with 2-mercaptoethanol for the second dimension (i.e. it appears as two spots in the second dimension), suggesting that NaTrxh only partially reduced S105-RNase before the first dimension. The specificity of NaTrxh reduction is apparent from comparison of the large number of proteins that shift mobility and appear off the diagonal after treatment with NaTrxh but not with the nonspecific reduction by DTT (compare Fig. 6, C and E). PAO affinity chromatography provided further evidence that S105-RNase is a substrate for NaTrxh. PAO matrices specifically bind proteins with vicinal thiols that can reversibly form a covalent bond with the resin (38Kalef E. Walfish P.G. Gitler C. Anal. Biochem. 1993; 212: 325-334Crossref PubMed Scopus (43) Google Scholar). Low salt extracts from SI N. alata S105S105 either were applied directly to the PAO matrix or were first treated with 20 mm 2-mercaptoethanol or NaTrxhrec plus NADPH and E. coli NTR. Fig. 7 shows that little or no S105-RNase bound to the PAO matrix unless it was first subjected to reducing conditions. Some S105-RNase was weakly bound under all conditions and eluted with 1 mm or 5 mm 2-mercaptoethanol (Fig. 7A). Large amounts of S105-RNase, however, bound tightly to this column after reduction by 2-mercaptoethanol (Fig. 7B) or NaTrxhrec (Fig. 7C). The tightly bound S105-RNase eluted with 500 mm 2-mercaptoethanol (Fig. 7, B and C). S105-RNase Interacts with NaTrx" @default.
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• W1991444355 date "2006-02-01" @default.
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• W1991444355 title "A Novel Thioredoxin h Is Secreted in Nicotiana alata and Reduces S-RNase in Vitro" @default.
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