Identification, cloning and characterization of ... - Wiley Online Library

Eur. J. Biochem. 270, 2633–2643 (2003) FEBS 2003

doi:10.1046/j.1432-1033.2003.03637.x

Identification, cloning and characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome Kenji Maeda, Christine Finnie, Ole Østergaard and Birte Svensson Department of Chemistry, Carlsberg Laboratory, Copenhagen, Denmark

Two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, were identified in two and one spots, respectively, in a proteome analysis of barley (Hordeum vulgare) seeds based on 2D gel electrophoresis and MS. HvTrxh1 was observed in 2D gel patterns of endosperm, aleurone layer and embryo of mature barley seeds, and HvTrxh2 was present mainly in the embryo. During germination, HvTrxh2 decreased in abundance and HvTrxh1 decreased in the aleurone layer and endosperm but remained at high levels in the embryo. On the basis of MS identification of the two isoforms, expressed sequence tag sequences were identified, and cDNAs encoding HvTrxh1 and HvTrxh2 were cloned by RT-PCR. The sequences were 51% identical, but showed higer similarity to thioredoxin h isoforms from other cereals, e.g. rice Trxh (74% identical with HvTrxh1) and wheat TrxTa (90% identical with HvTrxh2). Recombinant HvTrxh1, HvTrxh2 and TrxTa were produced in Escherichia coli and purified

using a three-step procedure. The activity of the purified recombinant thioredoxin h isoforms was demonstrated using insulin and barley a-amylase/subtilisin inhibitor as substrates. HvTrxh1 and HvTrxh2 were also efficiently reduced by Arabidopsis thaliana NADP-dependent thioredoxin reductase (NTR). The biochemical properties of HvTrxh2 and TrxTa were similar, whereas HvTrxh1 had higher insulin-reducing activity and was a better substrate for Arabidopsis NTR than HvTrxh2, with a Km of 13 lM compared with 44 lM for HvTrxh2. Thus, barley seeds contain two distinct thioredoxin h isoforms which differ in temporal and spatial distribution and kinetic properties, suggesting that they may have different physiological roles.

Thioredoxins are protein disulfide reductases of molecular mass 12 kDa [1]. The conserved active-site sequence WCGPC forms a disulfide bond in the oxidized form of the protein. The reduced, dithiol form of thioredoxin can modulate the activity of a variety of target proteins by reduction of their disulfide bonds. Plants contain several forms of thioredoxin which differ in their subcellular localization and thus in the target proteins with which they interact [2,3]. Thioredoxins f and m are found in chloroplasts and are involved in regulation of photosynthetic enzymes [4], whereas thioredoxin h is primarily cytosolic, and has also been identified in rice as one of the major protein components of phloem sap [5]. A mitochondrial thioredoxin system has been identified in Arabidopsis [6].

The catalytic system comprises, in addition to thioredoxin, an electron donor and a thioredoxin reductase, which are required for regeneration of the reduced form of thioredoxin. Thioredoxins f and m are reduced by ferredoxin via ferredoxin-dependent thioredoxin reductase [2]. Thioredoxin h is reduced by NADPH via NADP-dependent thioredoxin reductase (NTR) [7]. Thioredoxin h has been found to have an important influence on seed germination in barley and other plants [8,9]. Among the known target proteins of thioredoxin h in seeds are storage proteins such as hordeins in barley and glutenins and gliadins in wheat, which are deposited in disulfide-bound complexes and are mobilized during the germination process. Reduction by thioredoxin renders them more soluble and susceptible to proteolytic degradation [10]. Some a-amylase/trypsin inhibitor proteins [11] are also known targets of thioredoxin h, as is the barley a-amylase/subtilisin inhibitor (BASI) [12]. Studies of germinating transgenic barley seeds overexpressing wheat thioredoxin h revealed that limit dextrinase activity was increased [13], and that a-amylase activity increased earlier than in normal seeds [14]. This implicates thioredoxin h in regulation of the mobilization of starch reserves during seed germination. Multiple forms of thioredoxin h exist in plants; at least five isoforms have been identified in Arabidopsis [15] and three in wheat [16,17]. Whether these isoforms have different specificities or functions in the plant is not known. However, yeast complementation studies have provided evidence for different target specificities of Arabidopsis

Correspondence to C. Finnie, Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark. Fax: + 45 33274708, Tel.: + 45 33275304, E-mail: [email protected] Abbreviations: BASI, barley a-amylase/subtilisin inhibitor; DTNB, 5,5¢-dithio-bis-(2-nitrobenzoic acid); EST, expressed sequence tag; NTR, NADP-dependent thioredoxin reductase; TC, tentative consensus; TIGR, The Institute for Genomic Research. Proteins and enzymes: Arabidopsis NADP-dependent thioredoxin reductase (Q39243) (EC 1.8.1.9); barley a-amylase/subtilisin inhibitor (P07596); wheat thioredoxin h TrxTa (O64394); barley thioredoxin h1 HvTrxh1 (AY245454); barley thioredoxin h2 HvTrxh2 (AY245455). (Received 10 March 2003, revised 23 April 2003, accepted 28 April 2003)

Keywords: barley seed; disulfide reduction; proteomics; recombinant proteins; thioredoxin h.

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2634 K. Maeda et al. (Eur. J. Biochem. 270)

thioredoxin h isoforms [18]. Three forms of thioredoxin h have been observed in Western blots with barley seed extracts [9], and a gene has been identified in the wild barley Hordeum bulbosum that resembles a subset of thioredoxin h sequences [19]. However, until now, barley thioredoxin h has not been characterized at the protein level and target proteins in barley have been identified using thioredoxins from other organisms. Proteomics, based on the techniques of 2D gel electrophoresis and MS, offers the opportunity to study the appearance patterns of many proteins simultaneously, and to identify proteins of interest. Recently, these techniques have been applied to the study of seed development and germination in several plants including Arabidopsis [20,21], wheat [22] and barley [23–25]. We used 2D gel electrophoresis to identify thioredoxin h forms in barley seeds and characterize their patterns of appearance in the seed tissues and during germination. Identification of thioredoxin h forms in mature seed extracts by MS peptide mapping enabled cloning of the corresponding genes encoding two thioredoxin h forms with different appearance patterns. Recombinant proteins were produced and found also to differ in their biochemical properties. This is the first characterization of barley thioredoxin h proteins, and this comparative study of their properties extends our knowledge of the cereal thioredoxin h family.

Materials and methods Materials Rabbit antibody to wheat thioredoxin h was kindly supplied by B. Buchanan (UC Berkeley, CA, USA). Secondary antibodies were from Dako A/S. Primers were from DNA Technology (Aarhus, Denmark). Pfu DNA polymerase and BL21(DE3)Gold were from Stratagene. Restriction enzymes, DNA ligase and BL21(DE3)pLysS were from Promega. The pETtrxTa expression system was kindly provided by M. Gautier (INRA, Montpellier, France). Purified Arabidopsis thaliana NTR and Populus tremula thioredoxin h were kindly supplied by J.-P. Jacquot (INRA, Nancy, France). Bovine pancreas insulin, monobromobimane, isopropyl thio-b-D-galactoside, 5,5¢-dithiobis-(2nitrobenzoic acid) (DTNB) and NADPH were from Fluka. Plant material and protein extraction Spring barley (Hordeum vulgare cv. Barke) was field grown in Fyn, Denmark, in the 2000 season. Seeds were micromalted according to standard procedures [23]. Micromalted seeds were frozen in liquid nitrogen and freeze-dried before milling and extraction. Barley seeds were dissected as previously described [25]. Dissected tissues from five seeds were freeze-dried before extraction. Proteins were extracted from 4 g milled seeds in 20 mL extraction buffer (5 mM Tris/HCl, pH 7.5, 1 mM CaCl2) for 30 min at 4 C, as previously described [24]. Dissected tissues from five seeds were extracted in the same buffer for 30 min at 4 C as previously described [25]. After centrifugation to remove debris, supernatants containing soluble proteins were transferred to clean tubes and stored at )80 C until required.

2D gel electrophoresis Proteins contained in 250 lL and 100 lL mature seed extract were applied to the gels for colloidal Coomassie staining and 2D Western blotting, respectively. Duplicate gels were run containing proteins from 100 lL dissected seed extracts. By loading equal volumes, a similar ratio is seen between the proteins from each tissue on the dissected seed gels as the whole seed gels [25]. Germinated seed gels were also loaded with proteins from an equal volume of extract (100 lL), as the soluble protein content of germinated seeds is increased as the result of mobilized storage proteins. Thus, by loading equal volumes, proteins that remain at a constant level during germination will also appear at similar intensity on the 2D gels. Proteins were precipitated with 4 vol. acetone at )20 C for 24 h and resuspended in reswelling buffer [8 M urea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer 4–7 (Amersham Biosciences), 20 mM dithiothreitol and a trace of bromophenol blue]. IEF was carried out using 18 cm immobilized linear pH gradient (IPG) strips, pI 4–7, run on an IPGphor (Amersham Biosciences) as previously described [24]. Second-dimension SDS/polyacrylamide gels (12–14%, 18 · 24 cm; Amersham Biosciences) were run on a Pharmacia Multiphor II according to the manufacturer’s recommendations. Gels were stained with silver nitrate [26] or colloidal Coomassie blue [27]. For immunodetection of thioredoxin h, the 2D gel was blotted on to a nitrocellulose membrane in 10 mM CAPS, pH 11.0, using a Multiphor II NovaBlot unit (Amersham Biosciences). Immunodetection was carried out according to standard protocols, using a 1 : 2000 dilution of rabbit anti-(wheat thioredoxin h) serum. Goat anti-rabbit horseradish peroxidase-conjugated secondary IgGs were used at a 1 : 5000 dilution, and the signal was detected by enhanced chemiluminescence [28]. In-gel digestion and MALDI-TOF MS Spots were excised from the colloidal Coomassie-stained gel and subjected to in-gel trypsin digestion [29]. Tryptic peptides were desalted and concentrated on a home-made 5 mm nano-column [30] as previously described [24]. Peptides were eluted with 0.8 lL matrix (20 mgÆmL)1 a-cyano-4-hydroxycinnamic acid in 70% acetonitrile/0.1% trifluoroacetate) and deposited directly on to the MALDI target. A Bruker REFLEX III MALDI-TOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) in positive ion reflector mode was used to analyse tryptic peptides. The m/z software (Proteometrics, New York, NY, USA) was used to analyse spectra. Spectra were calibrated using trypsin autolysis products (m/z 842.51 and m/z 2211.10) as internal standards. To identify proteins, the SwissProt and NCBI nonredundant sequence databases and the NCBI expressed sequence tag (EST) databases were searched with peptide masses using the Mascot (http://www.matrix science.com) server. Tentative consensus (TC) sequences corresponding to identified EST sequences were obtained by searching the Institute for Genomic Research (TIGR) sequence database (www.tigr.org/tdb/tgi/hvgi).

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Barley seed thioredoxin h isoforms (Eur. J. Biochem. 270) 2635

Cloning and sequencing of barley thioredoxin h isoforms Embryos were dissected from five seeds after 24 h micromalting, and total RNA was extracted using the RNAeasy kit (Qiagen) according to the manufacturer’s recommendations, for use as a template in RT-PCR. Hvtrxh1. The coding sequence of HvTrxh1 was amplified by RT-PCR from barley embryo RNA using the primers trxh8 (TTCATATGGCCGCCGAGGAGGGAG) and trxh9 (GGGGATCCTAACCGGGCAATCACTCTTC). The primers were designed on the basis of the sequence of TC44851 and introduce an NdeI restriction site (underlined in trxh8) at the start codon (bold) and a BamHI restriction site (underlined in trxh9) after the stop codon. Reverse transcription and the following PCR was carried out on total RNA using an RT-PCR kit (Qiagen) and a PTC-200 Peltier Thermal Cycler (MJ Research). The RT-PCR product was cloned into pCR4-TOPO (Invitrogen) to give pCR-h1. Hvtrxh2. Primers trxh5 (TTGAATTCGCGTGAGAAA TAAGCCGAGT) and trxh6 (TTCTGCAGTCTTCTT GAGAGGACCTTTT), based on TC45680, were used for amplification of the HvTrx2 coding sequence as above. A second PCR with Pfu DNA polymerase and primers trxh1 (TTCATATGGCGGCGTCGGCAACGGCG) and trxh2 (GGGGATCCTGAGCGGCAATTTTATTTAGGCG) was used to introduce an NdeI restriction site (underlined in trxh1) at the start codon (bold) and a BamHI restriction site (underlined in trxh2) after the stop codon of HvTrxh2. The resulting PCR product was cloned into pCR Blunt II– TOPO (Invitrogen) to give pCR-h2. Construction of expression vectors. Inserts were isolated from pCR-h1 and pCR-h2 by digestion with Nde1 and BamHI and ligated into the pET11a expression vector linearized with Nde1 and BamHI, to give pETHvTrxh1 and pETHvTrxh2, respectively. The sequences of the inserts were determined on both strands and found to be as expected from the identified TC sequences, and confirmed that the cloning junctions were correct. Accession numbers for HvTrxh1 and HvTrxh2 sequences are AY245454 and AY245455, respectively. Expression and purification of recombinant barley thioredoxin isoforms Saturated cultures of Escherichia coli BL21(DE3)Gold transformed with pETHvTrxh1 or pETHvTrxh2 were diluted 100-fold in 2 L Luria–Bertani medium and grown at 37 C. After reaching an A600 of 0.6, the cultures were induced with 100 lM isopropyl thio-b-D-galactoside for 3 h. Cells were harvested and stored at )20 C until use. With the same procedure, wheat TrxTa was expressed in BL21(DE3)pLysS transformed with pETtrxTa [17]. HvTrxh1, HvTrxh2 and TrxTa were purified by a procedure for TrxTa [17] with minor modifications. Harvested cells were resuspended in 100 mL 50 mM Tris/HCl/ 1 mM EDTA, pH 8.0 and lysed by passage three times through a French Press. The lysate was sonicated to shear nucleic acids and centrifuged to remove insoluble material.

The supernantant was heat-treated for 7 min at 65 C and centrifuged to remove aggregated material. The supernatant was filtered and loaded on a HiLoad 26/10 Q Sepharose High Performance column (Amersham Biosciences) equilibrated with 30 mM Tris/HCl, pH 8.0. Proteins were eluted by a linear gradient from 0 to 700 mM NaCl in the same buffer. Thioredoxin h-containing fractions were detected by dot blotting 0.5 lL on to a nitrocellulose membrane. Immunodetection was carried out as described above, except that an alkaline phosphatase-conjugated swine antirabbit secondary IgG was used, and signal was detected using a 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium tablet (Sigma Chemical, St Louis, MO, USA). Thioredoxin-containing fractions were pooled and concentrated using a Centriprep YM3 (Millipore, Bedford, MA, USA) to 2 mL. The concentrated samples were then loaded on a HiLoad 16/60 Superdex 75 prep grad column (Amersham Biosciences) equilibrated with 30 mM Tris/HCl, pH 8.0, and eluted at a flow rate 0.2 mLÆmin)1. Thioredoxin-containing fractions were detected by dot-blotting as above and pooled. Purified proteins were quantified by amino-acid analysis. Amino-acid amounts in protein hydrolysates were determined using Biochrom 20 (Pharmacia Biotech). N-Terminal sequencing was performed using a 477A protein sequencer equipped with a 120A phenylthiohydantoin analyzer (Applied Biosystems). Liquid chromatography MS spectra of 100 pmol intact purified proteins were obtained using an HP 1100 LC/MSD (Hewlett-Packard). For SDS/PAGE, 3 lg HvTrxh1 or HvTrx2 was loaded on to a NuPAGE Bis/Tris 4–12% gel (Invitrogen) in the presence of 0.5 mM dithiothreitol. For native IEF, 1 lg HvTrxh1 or HvTrxh2 was loaded on to an IEF PhastGel covering the range pI 4.0–6.5 (Amersham Biosciences). Molecular modelling and sequence analysis The 3D structures of HvTrxh1 and HvTrxh2 were modelled using the SWISS-MODEL server (http://www.swissmodel. unibas.ch/) and Swiss PDB-viewer [31], with the crystal structure of thioredoxin h from Chlamydomonas reinhardtii (PDB accession 1EP7) [32] as a template. The modelled structures covered residues 5–115 and 11–121 of HvTrxh1 and HvTrxh2, respectively. The seqboot, protpars and consense programs in the PHYLIP 3.5 package [33] were used to generate an unrooted consensus tree based on an alignment of 46 plant thioredoxin h sequences from the NCBI sequence database. In addition to HvTrxh1 and HvTrxh2, sequences were from Arabidopsis thaliana (Ata, Q39241; Atb, NP_175128; Atc, NP_199112; Atd, S58119; Ate, NP_173403; Atf, Q8L907; Atg, Q39239; Ath, NP_190672; Ati, NP_188415), Brassica napus (Bna, Q42388; Bnb, Q39362), Brassica oleracea (Q9FQ63), Brassica rapa (Bra, O64432; Brb, Q8GZT3), Curcurbita maxima (Q8H9E2), Fagopyrum esculentum (Fe, Q96419), H. bulbosum (Hb, T50864), Hordeum vulgare (Hva, Q8GZR4), Leymus chinensis (Lc, AAO16555), Lolium perenne (Lp, T50865), Nicotiana tabacum (Nta, Q8H6 · 3); Ntb, Q07090; Ntc, P29449), Oryza sativa (Osa, Q42443; Osb, Q9FRT3; Osc, AAO37523; Osd, Q9AS75; Ose, Q8H6 · 4), Phalaris coerulescens (Pc, T50862), Picea mariana (Pm, O65049), Prunus persica (Pp, Q93WZ3),


Pisum sativum (Psa, Q8GUR8; Psb, Q9AR82; Psc, Q93 · 24; Psd, Q8GUR9), Populus tremula (Pt, Q8S3L3), Ricinus communis (Rc, Q43636), Secale cereale (Sc, T50863), Triticum aestivum (Taa, Q8GVD3; Tab, O64394; Tac, Q9LDX4; Tad, Q8H6 · 0) Triticum turgidum ssp. durum (Td, O64395) and Zea mays (Zm, Q8H6 · 5). SwissProt/ TREMBL accession numbers are given where available, otherwise EMBL/GenBank accession numbers are used. Enzyme assays Insulin assay [34]. A 1-mL reaction mixture contained 100 mM potassium phosphate, pH 7.0, 0.2 mM EDTA, 1 mg insulin, 1 lM HvTrxh1, HvTrxh2, or TrxTa, and 0.33 mM dithiothreitol. The reactions were initiated by addition of dithiothreitol. Reactions proceeded at room temperature (22 C) and were followed by measuring A650 on a Lambda 2 spectrophotometer (Perkin–Elmer). NTR assay [35]. A 200-lL reaction mixture contained 50 mM Tris/HCl, pH 8.0, 150 lM NADPH, 100 lM DTNB, 77 nM A. thaliana NTR [36], and 1–16 lM HvTrxh1, HvTrxh2, TrxTa, or Thioredoxin h-1 from P. tremulus [35]. Reactions proceeded at room temperature and were followed by measuring A405 on a MRX Revelation absorbance reader (Dynex Technologies). Reduction of BASI. A 50-lL reaction mixture contained 50 mM Tris/HCl, pH 7.5, 0.6 mM NADPH, 3.9 lg Arabidopsis NTR and either 10 lg recombinant BASI [37] or 10 lg insulin. The mixtures were incubated for 10 min at room temperature with 0.4 nmol HvTrxh1 or HvTrxh2. Free thiol groups were then labelled by the addition of 0.2 lmol monobromobimane in 10 lL acetonitrile. After incubation for 10 min at room temperature, proteins were precipitated with 80% (v/v) acetone before being loaded on a NuPAGE Bis-Tris 4–12% gel (Invitrogen). The labelled proteins were visualized by being photographed under near UV light.

Results Identification of thioredoxin h in the barley seed proteome To locate spots containing thioredoxin h in the barley seed proteome, a 2D gel with proteins extracted from mature barley seeds was electroblotted and probed with antibodies raised against wheat thioredoxin h. The antibody recognized three spots with an approximate molecular mass of 12 kDa and pI 5.0 (Fig. 1A). Protein spots excised from a colloidal Coomassie-stained gel of mature barley seed proteins were digested with trypsin and analysed by peptide mass mapping using MALDI-TOF MS. From the position of the spots recognized by the antibody, spots 296, 297 and 313 (Fig. 1A) were considered likely to contain thioredoxin h. The approximate molecular mass and pI of the proteins in these spots were determined from their positions on the 2D gel to be 12.2 kDa/pI 5.0, 12.5 kDa/pI 5.0 and 11.3 kDa/pI 5.0, respectively. Peptide mass data obtained for these spots did not lead to identifications in searches of the NCBI or SwissProt

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nonredundant databases, suggesting that the sequence data for these proteins were not present. However, by searching the NCBI EST sequence database, matches were obtained against barley EST accessions BE230983 (both spots 296 and 297) and BF626734 (spot 313). BLAST searches using these EST sequences indicated that both encoded proteins with homology to thioredoxin h. As ESTs can contain sequence errors and may not be fulllength, the matched EST sequences were used to search the TIGR database of TC sequences from barley. TC sequences were identified that contained the EST sequences and apparently encoded full-length proteins. This was supported by the fact that in-frame stop codons were present upstream of the ATG start codon in each case. The thioredoxin isoforms predicted to be encoded by these sequences were designated HvTrxh1 (TC44851; corresponding to spots 296 and 297) and HvTrxh2 (TC45680; spot 313). Theoretical tryptic digests of these sequences were used to determine the sequence coverage obtained from peptide mass mapping of the three spots. Peptide mass data from spot 313 resulted in 39% sequence coverage with seven matched peptides (Fig. 2A). Another TC sequence (TC45681) also matched the peptide masses from spot 313; this encoded a single amino-acid substitution at the C-terminus of the protein (A119 to G). As the peptide covering this region was not observed in the mass spectrum, it was not possible to distinguish between these almost identical variants. The peptide maps obtained for spots 296 and 297 were highly similar. The peptides matching the barley thioredoxin h TC sequence were the same in both cases, resulting in 64% sequence coverage with 12 matched peptides (Fig. 2A). It was therefore not possible from the peptide mass data to explain the molecular mass difference between the two spots on the 2D gel. Although the same EST sequence was matched for both spots, it is possible that spots 296 and 297 contain thioredoxin h isoforms with sequence differences in regions not covered by the peptide maps. However, no other barley TC sequences were found in the TIGR database that matched the peptide mass data for these spots. A few peptides originating from another protein (barley dimeric a-amylase inhibitor; P13691) were present in the spectrum obtained for spot 297 and in smaller amounts in the spectrum for spot 296. A spot containing this protein (O. Østergaard, C. Finnie, S. Melchior, P. Roepstorff & B. Svensson, unpublished results) forms a horizontal smear overlapping with spot 297 (Fig. 1A, spot 318; the smear is particularly noticeable in the silverstained gel), and this electrophoretic smear is probably the source of the peptides of barley dimeric a-amylase inhibitor. However, most of the protein in spots 297 and 296 was found to be thioredoxin h. Previously, evidence has been presented for variation in the distribution of thioredoxin h forms in seed tissues [9,16]. To obtain more detailed information about the origins of HvTrxh1 and HvTrxh2, the occurrence of the thioredoxin h spots in protein extracts from dissected barley seeds was analysed by 2D gel electrophoresis (Fig. 1B). The three thioredoxin h spots are distributed differently in the tissues of mature seeds (Fig. 1B). Spot 297 is present in extracts made from dissected endosperm, aleurone layer and embryo. Spot 296 is present in

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Coomassie 297 296 313

Silver 318

B

AL

ES

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Seed 3d

Seed 6d

EM 6d

Fig. 1. Barley thioredoxin h forms visualized on 2D gels. Sections of 2D gels from 11 to 16 kDa and pI 4.85–5.25 are shown. The positions of Trx h spots are indicated by circles. (A) Identification of Trx h in seed extracts by Western blotting. Corresponding spots 296, 297 and 313 on a colloidal Coomassie-stained gel were confirmed by MS to contain Trx h, and were also observed by silver staining. Spot 318 contains barley dimeric a-amylase inhibitor BDAI-1. (B) Tissue distribution of Trx h forms analysed using extracts from dissected barley seeds. AL, Aleurone layer; ES, starchy endosperm; EM, embryo. Proteins are visualized by silver staining. (C) Fate of Trx h forms during micromalting analysed using extracts from whole seeds after 3 and 6 days micromalting, and from embryo (EM) after 6 days micromalting. Proteins are visualized by silver staining.

endosperm and embryo, but is much less abundant in aleurone layer extracts. Spot 313 (containing HvTrxh2) is more abundant in extracts from the embryo than from the other tissues. In mature seeds, all three forms of thioredoxin h were observed on silver-stained 2D gels (Fig. 1A). Extracts from whole seeds made during micromalting showed that spots 297 and 313 were decreased in abundance after 3 and 6 days of germination, whereas spot 296, containing HvTrxh1, remained at a high level even after 6 days (Fig. 1C). Analysis of dissected seed extracts made after 6 days of micromalting showed that spot 296 remained abundant in the embryo (Fig. 1C) but was not detectable either in the aleurone layer or endosperm (not shown). The total amount of thioredoxin h has been shown to increase in the embryo and decrease in the endosperm during germination [9], in agreement with these observations.

Cloning and sequence analysis of barley thioredoxin h isoforms Based on the identified EST sequences, specific primers were designed for cloning of the two thioredoxin h isoforms. Both transcripts were isolated by RT-PCR using RNA isolated from barley embryos after one day of germination. Nucleotide sequencing demonstrated that the isolated clones were identical with the TC sequences for HvTrxh1 and HvTrxh2 identified on the basis of peptide mass data. The predicted amino-acid sequences of the proteins were 51% identical. The two thioredoxins were more similar to thioredoxin h sequences from other plants than to each other (Fig. 2A), as is also the case for Arabidopsis thioredoxins h [15]. HvTrxh1 was 74% identical with a thioredoxin h identified in rice as an abundant phloem sap protein (Q42443) [5]. HvTrxh2 was 53% identical with this

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HvTrxh2 Ta O64394 Os Q9FRT3 HvTrxh1 Os Q42443

MAAS_____ATAAAVAA_EVISVHSLEQWTMQIEEANTAKKLVVIDFTASWCGPCRIMAP ----AATAT-----VG-G-------------------A------------------------AS____---Q-EGT__--AI---DE--I------S-------------------I-__________M--EEG-__--AC-TKQEFDTHMANGKDTG---I------------VI-__________M--EEGV__--AC-NKDEFDA-MTK-KE-G-V-I------------FI--

54 60 54 48 48

VFADLAKKFPNAVFLKVDVDELKPIAEQFSVEAMPTFLFMKEGDVKDRVVGAIKEELTAKVGLHAAAQ______ I---------A-----------------------------------------------T-------Q_______ --------HT------------------------------------------M-D—-AS-LE—-M-________ ---EY-----G-I----------DV--AYN---------I-D-EKV-S---GR-DDIHT-IVALMGSAST____ ---EY-----G------------EV--KYN---------I-D-AEA-K----R-DD-QNTIVK-VGATAASASA

B

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Lc

Hb

Pc

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Zm Ose Osd

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122 127 120 118 122

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Osc Pm

Ati Cm

R Taa HvTrxh1 Osa

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I/M Tab Tac

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Osb

Pt Rc Psb Ath Ntb

HvTrxh2 Ata Atb Atc

A

Bnb Bra Bo

K

Atg Atf Atd Ate

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Bna

Fig. 2. Sequence alignment of plant thioredoxin h sequences (A), modelled structure of barley HvTrxh1 (B), using the structure of C. reinhardtii Trx h (PDB accession 1EP7) as a template, and consensus tree of 46 plant thioredoxin h sequences (C), using the alignment region delineated by vertical lines in (A). (A) Barley HvTrxh1 and HvTrxh2 sequences aligned with TrxTa from wheat (Ta, O64394) and two Trxh sequences from rice (Os, Q9FRT3 and Q42443). Only residues differing between sequences are shown. Dashes indicate identity between sequences and underscores indicate gaps introduced into the alignment. Peptides observed in mass spectra for spots 297 (HvTrxh2) and 313 (HvTrxh1) are boxed. The conserved active-site sequence is in bold. The residues corresponding to R101 in HvTrxh1 are marked by an arrow. Vertical lines delineate the region used for the analysis in (C). (B) Residues differing in HvTrxh1 and HvTrxh2 are shaded. Side chains are shown for the active-site cytseines and R101, which is replaced by isoleucine in HvTrxh2. (C) HvTrxh1 and HvTrxh2 are indicated. Cereal sequences are in bold and indicated by filled circles. Species are identified by initials and isoforms by italicised suffix. Accession numbers are given in Materials and methods. The identity of the residue corresponding to R101 in HvTrxh1 is given for each of the circled clusters. The cluster marked with an asterisk corresponds to the previously described subgroup of thioredoxin h sequences [19].

protein, but 90% identical with wheat TrxTa (O64394) [17] and 78% identical with rice rTrxh2 (Q9FRT3) [38]. HvTrxh1 shared only 49% and 53% identity, respectively, with these proteins. The TIGR database contains additional barley sequences (TC44856 and TC56664) encoding thioredoxin h. These thioredoxin h forms have an elongated N-terminal sequence, and TC56664 is very similar to the H. bulbosum sequence (T50864) identified from mature pollen and

described as a member of a thioredoxin h subgroup [19]. The isoform encoded by TC44856 is 35% and 38% identical, respectively, with HvTrxh1 and HvTrxh2. The calculated molecular mass and pI for these predicted proteins are 14.5 kDa/pI 5.9 and 14.4 kDa/pI 5.2, respectively. No spots in this area, however, were observed on the 2D Western blot with the antibody to wheat thioredoxin h. This suggests that the antibody does not recognize these isoforms because several of the ESTs contained in the TC

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sequences originate from cDNA libraries from developing or germinating barley seeds. Alternatively, these isoforms may not be present in mature seeds. It is currently not known whether this subclass of thioredoxin h has a specific function [19]. As no structure is yet available for plant thioredoxin h, the structures of HvTrxh1 and HvTrxh2 were modelled, using the crystal structure of C. reinhardtii thioredoxin h [32] as a template (Fig. 2B). The structure of C. reinhardtii thioredoxin h is highly superimposable on that of E. coli thioredoxin, despite only limited sequence identity. Most of the sequence variation between HvTrxh1 and HvTrxh2 occurs in the N-terminal and C-terminal parts. Thus, although HvTrxh1 and HvTrxh2 have an overall identity of 51%, residues 29–99 of HvTrxh1 and HvTrxh2 (HvTrxh1 numbering) are 75% identical. The residues differing in HvTrxh1 and HvTrxh2 were mapped on to the modelled structure of HvTrxh1 (Fig. 2B). These are mainly distributed away from the active site. The loops close to the active site consist nearly exclusively of conserved residues. The most significant difference between the two isoforms in this region is an arginine at residue 101 of HvTrxh1, located in the loop before the C-terminal a-helix. This residue is replaced by isoleucine in HvTrxh2 (Figs 2A.B), which would lead to a local charge difference on the surface of the proteins 12 A˚ from the active-site disulfide bridge. A comparison of the other related thioredoxin sequences (Fig. 2A) shows that the rice thioredoxin most similar to HvTrxh1 also has arginine at this position, whereas the sequences more similar to HvTrxh2 have isoleucine or methionine. This trend was supported by a more detailed analysis of 46 plant thioredoxin h sequences (Fig. 2C) in which cereal sequences cluster into different groups where the equivalent residue is R in HvTrxh1-like sequences, hydrophobic (I or M) in HvTrxh2-like sequences, and N in the thioredoxin h subgroup previously defined [19]. Noncereal thioredoxin h sequences formed additional clusters that also correlated with the identity of this residue (A, N or K; Fig. 2C).

A

B

kDa

pI 6.55

97.4 66.3 55.4

5.85

36.5 31.0 5.20 21.5 14.4 4.55 6.0

4.15

1

2

1

2

Fig. 3. Purified recombinant barley thioredoxin h. (A) SDS/polyacrylamide gel loaded with 3 lg purified recombinant thioredoxins. (B) Native IEF gel loaded with 1 lg purified recombinant thioredoxins. Lane 1, HvTrxh1; Lane 2, HvTrxh2. Gels are stained with Coomassie blue. Molecular mass and pI markers are indicated.

Expression vectors of recombinant barley thioredoxin h isoforms were constructed for production of nontagged fulllength thioredoxin h isoforms based on the previous expression and purification system developed for wheat thioredoxin h (TrxTa) [17]. HvTrxh1, HvTrxh2 and TrxTa were thus produced in E. coli, and both recombinant barley thioredoxin h isoforms were recognized by the antibody to wheat thioredoxin h. The proteins were purified as described in Materials and methods. The yields of purified proteins were 8 mgÆL)1 culture for HvTrxh1, 3 mgÆL)1 culture for HvTrxh2, and 1.5 mgÆL)1 culture for TrxTa. This compares well with the previously published yield of 5 mgÆL)1 for TrxTa produced from the same expression system [17].

HvTrxh1. This value was not in agreement with the predicted mass of the full-length protein (12 754.70 Da) but matched the predicted mass of the protein lacking the N-terminal methionine and with the active-site cysteines in oxidized form (12 621.49 Da). N-Terminal sequencing confirmed that the N-terminal methionine had been cleaved off. For HvTrxh2, two peaks were obtained by liquid chromatography MS. One peak (13 032.75 Da) matched the predicted mass of the protein lacking the N-terminal methionine and with oxidized active-site cysteines (13 033.17 Da). The second peak (12 961.53 Da) was 71 Da smaller, indicating that a fraction of the purified protein was missing methionine and alanine from the N-terminus. This was confirmed by the N-terminal sequences of HvTrxh2, determined to be AASATAAAVA and ASATAAAVAA. Multiple peaks were observed in the mass spectrum of TrxTa. The largest mass obtained was 12 675.91 Da, matching the predicted mass of TrxTa with oxidized active-site cysteines and lacking 10 residues at the N-terminus (12 675.80 Da). Additional values of 12 574.22 Da, 12 503.50 Da and 12 432.42 Da were observed that corresponded to cleavage of the N-terminal segment at residues 11, 12 and 13, respectively. SDS/PAGE and IEF supported the purity of the recombinant proteins. In SDS/PAGE, HvTrxh1 and HvTrxh2 had apparent molecular masses of 10 kDa and 9 kDa, respectively (Fig. 3A). These values are slightly lower than the predicted or MS determined masses and the apparent molecular masses of the thioredoxin h isoforms as observed in 2D gels of barley protein extracts. Isoelectric points of 5.2 for HvTrxh1 (predicted pI 5.09) and 5.3 for HvTrxh2 (predicted pI 5.2) were obtained by native IEF (Fig. 3B). Faint bands were observed at slightly lower pI in both cases.

Characterization of purified proteins

Activity of thioredoxin h isoforms

Masses of the recombinant proteins were obtained using liquid chromatography MS. A single peak corresponding to an average mass of 12 621.23 Da was obtained for purified

Insulin reduction. The recombinant barley thioredoxin h isoforms were analysed for their ability to reduce insulin [34] and compared with the wheat thioredoxin TrxTa (Fig. 4A).

Expression and purification of recombinant thioredoxin h

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Reaction mixtures containing 1 lM TrxTa, HvTrxh1 or HvTrxh2 showed faster aggregation of insulin compared with a control containing only dithiothreitol, demonstrating that all three recombinant proteins could catalyse insulin reduction. The activity of HvTrxh1 under these conditions

A

2.0

TrxTa HvTrxh1 HvTrxh2 control

A650

1.6 1.2 0.8 0.4 0 0

10

20

30

40

time (min)

B V (∆A405/min)

0.10

TrxTa HvTrxh1 HvTrxh2

0.08 0.06 0.04

was 1.7 A650Æs)1Ælmol)1, whereas HvTrxh2 and TrxTa were more similar to each other, with activities of 1.3 and 1.2 A650Æs)1Ælmol)1, respectively. Reduction of thioredoxin h by NTR. Recombinant HvTrxh1, HvTrxh2 and TrxTa were tested and compared as substrates for NTR from A. thaliana [36] (Fig. 4B). The reduction of thioredoxin h was followed by measuring the increase in A405 caused by reduction of DTNB [35]. A. thaliana NTR was able to reduce all three thioredoxin h proteins. The resulting Vmax and Km values for HvTrxh1 were 2.5 nmolÆs)1Ænmol)1 and 13 lM. Again, HvTrxh2 and TrxTa resembled each other with Vmax of 2.0 and 1.5 nmolÆs)1Ænmol)1 and Km of 44 lM and 37 lM, respectively. In the same assay, a Km value of 8 lM was obtained for thioredoxin h from P. tremulus (data not shown). For comparison, a Km value of 1.5 lM was reported for this protein under similar assay conditions [35]. The slight discrepancy between the two studies may be explained by slight differences in the analysis of the amounts of protein applied as amino-acid analysis was used for calculation of the protein concentration in the present study. Reduction of BASI. Previously, BASI has been identified as a possible target of thioredoxin h, on the basis of its reduction by recombinant wheat thioredoxin h [12]. To determine whether both endogenous thioredoxins were able to reduce BASI, purified recombinant BASI [37] was incubated with HvTrxh1 or HvTrxh2 in the presence of Arabidopsis NTR and NADPH. The reduced cysteines in BASI were subsequently fluorescence labelled with monobromobimane. The proteins were separated by SDS/PAGE and the labelled products were visualized under UV light (Fig. 4C). Both HvTrxh1 and HvTrxh2 were able to reduce BASI and insulin in this system.

Discussion

0.02 0 0

5

10

15

[trxh] (µM)

C NTR BASI Trxh Insulin 1

2

3

4

5

20

Between one and three bands in 1D Western blots of protein extracts from barley seed tissues were recognized by the antibody to wheat thioredoxin h [9]. The question was raised whether these bands represented different thioredoxin h isoforms. In the present study using 2D Western blotting, three spots were also observed and these were used to locate thioredoxin h-containing spots on Coomassiestained and silver-stained 2D gels. Owing to the limited amount of barley sequence information in the databases, it was necessary to use EST sequence data for identification of Fig. 4. Activity measurements of thioredoxin h. (A) Time course of insulin reduction by purified recombinant thioredoxins. (B) Reduction of recombinant thioredoxins by Arabidopsis NTR, monitored via the reduction of DTNB. (h) HvTrxh1; (s) HvTrxh2; (n) TrxTa; (·) control (without addition of thioredoxin). (C) Reduction of BASI and insulin by barley thioredoxin h isoforms. BASI (lanes 1–3) or insulin (lanes 4 and 5) were incubated with NTR and NADPH without the addition of thioredoxin h (lane 1) or together with HvTrxh1 (lanes 2 and 4) or HvTrx2 (lanes 3 and 5). Free thiols were labelled with monobromobimane, and reaction mixtures were run on SDS/PAGE and visualized under UV. The positions of NTR, BASI, thioredoxin and insulin bands are indicated.

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these spots. This led to the identification of two new thioredoxin h isoforms, designated HvTrxh1 and HvTrxh2. From MALDI-TOF peptide mass mapping data, it is probable that the two spots of higher molecular mass contain the same isoform (HvTrxh1), whereas the spot of lower molecular mass contains HvTrxh2. Further analysis, involving more advanced MS techniques, will be required to explain the appearance of HvTrxh1 in two spots. Silver-stained 2D gels with extracts from the aleurone layer, endosperm and embryo show in detail how HvTrxh1 and HvTrxh2 are distributed in these tissues. HvTrxh1 from all three tissues is observed. The relative intensity of the two HvTrxh1 spots from the different tissues varies (Fig. 1), suggesting that the degree of modification giving rise to the two spots differs among these tissues. HvTrxh2 shows a different distribution to HvTrx1, being most abundant in extracts from embryo. Thioredoxin h has previously been reported to decrease in endosperm and increase slightly in the embryo in wheat and barley during germination [9,16]. This study confirms these observations, but demonstrates that the two barley isoforms display different temporal patterns of appearance. Whereas HvTrx2 decreases in abundance during micromalting, and HvTrxh1 also decreases in abundance in the aleurone layer and endosperm, the lower form of HvTrxh1 remains at a high level in germinated embryo. These results suggest that the two isoforms are differentially regulated in the seed tissues and may have different physiological roles. Higher plants are characterized by having many thioredoxin h isoforms [3,15], typically with divergent sequences that share higher sequence identity between than within species. The two barley isoforms identified here are also more similar to other cereal thioredoxin h isoforms than to each other, and at least two other barley thioredoxin h sequences with low identity with HvTrxh1 and HvTrxh2 are present in the TIGR barley sequence database. As only HvTrxh1 and HvTrxh2 have been identified so far in the barley seed proteome, the other thioredoxin h isoforms may not be present in high abundance in mature seeds. The question remains whether this sequence diversity of thioredoxin h isoforms in barley and other plants reflects differences in their biochemical properties or physiological roles. The coding sequences for HvTrxh1 and HvTrxh2 were successfully cloned on the basis of the EST sequences identified by peptide mass mapping of spots on 2D gels. The purification procedure developed for TrxTa [17] was also suitable for HvTrxh1 and HvTrxh2. Both isoforms were confirmed to have protein disulfide reductase activity using insulin as a substrate, and both were efficiently reduced by Arabidopsis NTR, as might be expected from the high sequence identity between NTR from Arabidopsis and wheat [39]. TrxTa and HvTrxh2, which share the highest sequence identity, showed very similar properties in both assays. The properties of HvTrxh1 and HvTrxh2 were, however, more divergent. Under the conditions used, HvTrxh1 was both more active in insulin reduction and a better substrate for Arabidopsis NTR (with threefold lower Km). Thioredoxins h1 and h2 from P. tremula (with 31% sequence identity) are also reported to have different biochemical properties. The Km of A. thaliana NTR was 1.5 and 12 lM for P. tremula thioredoxin h1 and h2, respectively [35,40].

The structure/function relationships will need to be analysed to understand the differences in biochemical properties between HvTrxh1 and HvTrxh2. The residues that differ between HvTrxh1 and HvTrxh2 are mainly at the N-termini and C-termini of the proteins, and they are mostly located away from the active site in the modelled 3D structure. However, R101 of HvTrxh1, replaced by I105 in HvTrxh2, is located 12 A˚ from the active site. This charge difference on the surface of the protein may influence the interaction with target proteins. Heterologous expression of chimeric Arabidopsis thioredoxin h isoforms in yeast suggested that the residues necessary for interaction with different targets are located in different regions of the proteins [41]. Interestingly, the N-terminal sequence of HvTrxh1 contains the sequence MAAEE (Fig. 2), which has been suggested in the rice phloem sap thioredoxin h (accession Q42443) to be involved in phloem trafficking of the protein [38]. By alanine scanning, a four-amino-acid region of the rice protein containing arginine at the equivalent position to R101 in HvTrxh1 was also found to affect phloem trafficking [38]. Mutagenesis studies will be required to determine the importance of this and other residues in HvTrxh1 and HvTrxh2. Identification of target proteins of thioredoxin h has been reported in several plants including peanuts [42] and barley embryo [9]. As barley thioredoxin h has not been available until now, target proteins of thioredoxin h in barley, including BASI [12], have previously been identified using heterologous thioredoxin h. It is now demonstrated here that BASI can be reduced by both endogenous thioredoxin h isoforms. Heterologous expression of Arabidopsis thioredoxin h isoforms in yeast showed that different thioredoxin h isoforms can interact with different proteins [18]. Further work is required to determine whether the different properties of barley HvTrxh1 and HvTrxh2 are reflected in different target specificities. In conclusion, the identified isoforms of barley thioredoxin h, HvTrxh1 and HvTrxh2, show differences in tissue distribution, temporal appearance, and biochemical properties. Further characterization of both proteins and identification of specific interaction partners will contribute to a fuller understanding of the functions of thioredoxin h in barley seeds.

Acknowledgements Mette Hersom Bien, Sidsel Ehlers, Lone H. Sørensen and Pia Breddam are gratefully acknowledged for technical assistance, Sejet Plantbreeding for seed material and Jørgen Larsen and Ella Meiling (Carlsberg Research Laboratory) for micromalted samples. We thank Professor B. Buchanan (UC Berkeley) for rabbit anti-(wheat thioredoxin h) IgG, Dr M. Gautier (INRA, Montpellier, France) for pETtrxTa expression system, and Professor J.-P. Jacquot (INRA, Nancy, France) for purified A. thaliana NTR, and P. tremula thioredoxin h, B. C. Bønsager (Carlsberg Laboratory) for recombinant BASI, and Karen Skriver (University of Copenhagen) and Kristian Sass Bak-Jensen (Carlsberg laboratory) for helpful discussions. K.M. is supported by a scholarship from Carlsbergs Mindelegat for Brygger J. C. Jacobsen. O.Ø. is supported by a Ph.D. fellowship (no. EF803) from the Danish Academy of Technical Sciences. The project is supported by the Danish Research Agency’s SUE programme (samarbejde mellem sektorforskning, universitet og erhverv) grant no. 9901194.


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