Báo cáo khoa học: Identification, cloning and characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome pot

Identification, cloning and characterization of two thioredoxinh isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome Kenji Maeda, Christine Finnie, Ole Østergaard and Birte Sven

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

abun-dance and HvTrxh1 decreased in the aleurone layer and

endosperm but remained at high levels in the embryo On the

basis of MSidentification of the two isoforms, expressed

sequence tag sequences were identified, and cDNAs

enco-ding 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 thio-redoxin 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 Kmof 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 proper-ties, suggesting that they may have different physiological roles

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

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

chloro-plasts 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 thio-redoxin Thioredoxins f and m are reduced by ferredoxin via ferredoxin-dependent thioredoxin reductase [2] Thio-redoxin 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 degrada-tion [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 germi-nating transgenic barley seeds overexpressing wheat thio-redoxin 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: csf@crc.dk

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)

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 bulbosumthat 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

electro-phoresis 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

electropho-resis 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 MSpeptide 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

know-ledge of the cereal thioredoxin h family

Materials and methods

Materials

Rabbit antibody to wheat thioredoxin h was kindly

sup-plied 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)pLysSwere 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,

monobromo-bimane, isopropyl thio-b-D-galactoside,

5,5¢-dithiobis-(2-nitrobenzoic 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

micro-malted according to standard procedures [23] Micromicro-malted

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 4C, as previously described [24]

Dissected tissues from five seeds were extracted in the same

buffer for 30 min at 4C 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] Germi-nated 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 [8Murea, 2% (w/v) CHAPS, 0.5% (v/v) IPG buffer 4–7 (Amersham Biosciences), 20 mM dithiothreitol and a trace of bromo-phenol 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 Phar-macia 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 mMCAPS,

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 horse-radish 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)

Cloning and sequencing of barley thioredoxin h isoforms

Embryos were dissected from five seeds after 24 h

micro-malting, and total RNA was extracted using the RNAeasy

kit (Qiagen) according to the manufacturer’s

recommenda-tions, 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

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

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 37C After reaching an A600of 0.6, the cultures were

induced with 100 lMisopropyl 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)pLysStransformed with pETtrxTa [17]

HvTrxh1, HvTrxh2 and TrxTa were purified by a

procedure for TrxTa [17] with minor modifications

Har-vested cells were resuspended in 100 mL 50 mMTris/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 65C and centrifuged to remove aggregated material The supernatant was filtered and loaded on a HiLoad 26/10 Q Sepharose High Performance column (Amersham Biosciences) equi-librated with 30 mMTris/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 anti-rabbit 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 concen-trated 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 mMTris/HCl,

pH 8.0, and eluted at a flow rate 0.2 mLÆmin)1 Thio-redoxin-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 deter-mined using Biochrom 20 (Pharmacia Biotech)

N-Terminal sequencing was performed using a 477A protein sequencer equipped with a 120A phenylthiohydan-toin analyzer (Applied Biosystems) Liquid chromatogra-phy MSspectra 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

PHYLIP3.5 package [33] were used to generate an unrooted consensus tree based on an alignment of 46 plant thio-redoxin 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, Q9AS 75; 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 (22C) 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 A405on 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 mMNADPH, 3.9 lg

Arabi-dopsis 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

recog-nized 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

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).BLASTsearches using these EST sequences indicated that both encoded proteins with homology to thioredoxin h

As ESTs can contain sequence errors and may not be full-length, 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 silver-stained 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

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 redoxins h [15] HvTrxh1 was 74% identical with a thio-redoxin h identified in rice as an abundant phloem sap protein (Q42443) [5] HvTrxh2 was 53% identical with this

ES

B

Seed 6d

C

297 296 313

318

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 MSto 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.

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, respect-ively 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

HvTrxh2 MAAS _ATAAAVAA_EVISVHSLEQWTMQIEEANTAKKLVVIDFTASWCGPCRIMAP 54

Ta O64394 AATAT -VG-G -A - 60

Os Q9FRT3 -AS -Q-EGT AI -DE I -S -I 54

HvTrxh1 M EEG- AC-TKQEFDTHMANGKDTG -I -VI 48

Os Q42443 M EEGV AC-NKDEFDA-MTK-KE-G-V-I -FI 48

VFADLAKKFPNAVFLKVDVDELKPIAEQFSVEAMPTFLFMKEGDVKDRVVGAIKEELTAKVGLHAAAQ 122 I -A -T -Q _ 127 -HT -M-D—-AS-LE—-M- 120 -EY -G-I -DV AYN -I-D-EKV-S -GR-DDIHT-IVALMGSAST 118 -EY -G -EV KYN -I-D-AEA-K R-DD-QNTIVK-VGATAASASA 122

A

Active site

R101

B

N

R

I/M

HvTrxh1

HvTrxh2

Taa

Osa

Osb

Td

Tab Tac

Tad Hva

Sc

Lp Hb

Osc

C

K

N A

Zm

Ose Osd

Ata AtbAtc

AtdAte Atf Atg Bna

Bo

Bnb Bra

Brb

Pm

Fe Cm

Nta

Pt

Ath

Ati

Rc

Ntb

Ntc

Pp

Psa

Psb

Psc Psd

*

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].

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]

Non-cereal thioredoxin h sequences formed additional clusters

that also correlated with the identity of this residue (A, N or

K; Fig 2C)

Expression and purification of recombinant thioredoxin h

Expression vectors of recombinant barley thioredoxin h

isoforms were constructed for production of nontagged

full-length 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

des-cribed 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]

Characterization of purified proteins

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

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 recom-binant proteins In SDS/PAGE, HvTrxh1 and HvTrxh2 had apparent molecular masses of 10 kDa and 9 kDa, respect-ively (Fig 3A) These values are slightly lower than the predicted or MSdetermined 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 Activity of thioredoxin h isoforms

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)

6.0 14.4 21.5 31.0 36.5 55.4 66.3 97.4

kDa

1 2

5.20

4.55 4.15

5.85 6.55

pI

1 2

Fig 3 Purified recombinant barley thioredoxin h (A) SDS/polyacryl-amide 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.

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

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 A405caused by reduction of DTNB [35]

A thalianaNTR was able to reduce all three thioredoxin h proteins The resulting Vmaxand Kmvalues for HvTrxh1 were 2.5 nmolÆs)1Ænmol)1and 13 lM Again, HvTrxh2 and TrxTa resembled each other with Vmax of 2.0 and 1.5 nmolÆs)1Ænmol)1and Kmof 44 lMand 37 lM, respect-ively In the same assay, a Kmvalue of 8 lMwas obtained for thioredoxin h from P tremulus (data not shown) For comparison, a Kmvalue of 1.5 lMwas 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 ArabidopsisNTR and NADPH The reduced cysteines in BASI were subsequently fluorescence labelled with mono-bromobimane 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

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 thio-redoxin 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 Coomassie-stained and silver-Coomassie-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

0

0.4

0.8

1.2

1.6

2.0

HvTrxh2 HvTrxh1 TrxTa

control

time (min)

A 65

A

0

0.02

0.04

0.06

0.08

0.10

[trxh] ( µM)

A 40

HvTrxh2 HvTrxh1 TrxTa B

Trxh

BASI

NTR

Insulin

1 2 3 4 5

C

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.

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 MStechniques, 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

decrea-ses 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

thio-redoxin 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 Kmof 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 Hetero-logous 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 thio-redoxin 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 thio-redoxin 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 Plantbreed-ing for seed material and Jørgen Larsen and Ella MeilPlantbreed-ing (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 sektorforsk-ning, universitet og erhverv) grant no 9901194.

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