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SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION

The NADPH-Dependent Thioredoxin Reductase/Thioredoxin System in Germinating Barley Seeds: Gene Expression, Protein Profiles, and Interactions between Isoforms of Thioredoxin h and Thioredoxin Reductase^1,[W]

Enzyme and Protein Chemistry, BioCentrum-DTU, Technical University of Denmark, DK–2800 Kgs. Lyngby, Denmark

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer.

NTRs belong to a superfamily of flavoprotein disulfide oxidoreductases (Reichheld et al., 2005) and transfer electrons from NADPH to the active-site disulfide bridge of oxidized Trx h via FAD and a redox-active disulfide (Mustacich and Powis, 2000). NTRs from plants, bacteria, fungi, and archaea are homodimers of 35 kD (Dai et al., 1996), whereas mammalian NTRs are homodimers of 55 kD, usually containing an active-site seleno-Cys (Tamura and Stadtman, 1996). The NTR/Trx system in plants has a variety of functions. Trx h was suggested to act as a signal in the phloem sap (Ishiwatari et al., 1995) and it was reported to participate in the self-incompatibility reaction (Cabrillac et al., 2001). In addition, Trx h functions during seed development (Serrato and Cejudo, 2003). Many of the Trx h target proteins identified in cereal seeds suggest that cellular redox-regulated processes are necessary for germination. Thus, the NTR/Trx system may control the activity of -amylase and trypsin inhibitors (Kobrehel et al., 1991), increase activity of pullulanase (limit dextrinase) that specifically cleaves -1,6 linkages from starch amylopectin (Cho et al., 1999), reduce storage proteins, facilitating their mobilization (Kobrehel et al., 1992), and activate a seed-specific Ser protease, thiocalsin (Besse et al., 1996). Finally, enhancement of gibberellic acid (GA) synthesis in embryos and accelerated appearance of -amylase in transgenic barley overexpressing Trx h in the starchy endosperm suggest that Trx h plays a role in the communication between endosperm, embryo, and aleurone layer (Wong et al., 2002).

The presence of multiple Trx h and NTR isoforms in plants makes the NTR/Trx system particularly complex compared with other organisms. For instance, in Arabidopsis (Arabidopsis thaliana), eight genes encoding Trx h have been identified (Meyer et al., 2002, 2005) and three NTR isoforms have been characterized (Serrato et al., 2004; Reichheld et al., 2005). Five Trx h (Gautier et al., 1998; Serrato et al., 2001; Cazalis et al., 2006) and one NTR (Serrato et al., 2002) were described from wheat (Triticum aestivum). In barley (Hordeum vulgare), two seed Trx h isoforms were characterized (Maeda et al., 2003), but no barley NTR was identified until now.

The mechanisms regulating expression of Trx h and NTR in seed tissues are poorly understood; indeed, specific isoforms of Trx h and NTR may be localized in different tissues and have diverse roles during plant development. Expression of Trx h in endosperm seems to be controlled by hormones via the embryo (Lozano et al., 1996). Furthermore, an increase of Trxh1, Trxh2, and Trxh3 transcripts at the beginning of seed desiccation and a transient increase of Trxh1 after seed imbibition were observed in wheat (Cazalis et al., 2006). Others reported that Trx h (Marx et al., 2003) and NTR (Montrichard et al., 2003) increase during germination in the embryo of barley and pea (Pisum sativum), respectively.

Knowledge lags behind on the individual roles played by Trx and NTR isoforms from the same organism. In particular, insight is lacking on specificity and structural requirements for interactions between NTRs and Trxs. To address these fundamental questions, two barley NTR isoforms are cloned and characterized in this work and their gene expression and protein appearance in seed tissues are described in parallel with two Trx h isoforms. The effects of GA and abscisic acid (ABA) on the members of this NTR/Trx system were monitored in isolated aleurone layers. Production of all four proteins in recombinant form allowed the interactions between isoforms of Trx h and NTR from the same source to be characterized.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Isolation, Cloning, and Sequence Analysis of Two cDNAs Encoding NTR

Two cDNAs encoding NTR in barley seeds were isolated by a PCR-based cloning strategy. Each contained an open reading frame of 996 bp encoding proteins designated HvNTR1 and HvNTR2 with theoretical molecular mass (kD)/pI of 34.818/5.7 and 34.793/5.7, respectively. Nucleotide sequences of HvNTR1 and HvNTR2 are very similar to tentative consensus (TC) sequences TC142091 and TC141301 from the The Institute for Genomic Research (TIGR) barley gene index. One additional sequence in the database (TC132362) showed lower identity with HvNTR1 and HvNTR2, but was still related to NTR. TCs were assembled from few EST sequences, indicating that NTR transcripts are not highly represented in barley cDNA libraries. It therefore cannot be excluded that barley has other NTR isoforms than the three discussed here. The EST sequences originated predominantly from seed tissues.

HvNTR1 and HvNTR2 proteins have 88% sequence identity. A multiple alignment, including the protein sequence deduced from TC132362 and NTR sequences from other sources (Supplemental Fig. S1), was used to generate a phylogenetic tree (Fig. 1 ). The tree is divided into three major clusters. One cluster contains NTR from cyanobacteria and plant chloroplast-type (C-type) NTRs that have an extra Trx active-site sequence (CGPC) in a C-terminal extension (Serrato et al., 2004; Alkhalfioui et al., 2007a). The second cluster has two main branches; one contains fungal NTRs and the other contains plant cytoplasmic or mitochondrial-type NTRs (A/B type; Reichheld et al., 2005), which are further subdivided into sequences from monocots and dicots. Bacterial NTRs constitute the third cluster. HvNTR1 and HvNTR2 belong to the monocotyledon subgroup of the plant A/B type. Despite their high sequence identity, both have even higher similarity to other cereal NTRs. HvNTR1 is thus 97% identical with wheat NTR (TaNTR) and HvNTR2 is 91% identical to a putative rice (Oryza sativa) NTR (Os1; see Fig. 1). HvNTR1 and HvNTR2 are each 75% identical to an Arabidopsis NTR (At3; Fig. 1). Barley enzymes are thus expected to share tertiary and quaternary structure with Arabidopsis NTR—the only plant NTR for which the three-dimensional (3-D) structure has been solved (Dai et al., 1996).

View larger version (24K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Phylogenetic tree of NTR sequences. Values indicate percent bootstrap support for each branch. For accession numbers and organisms, see "Materials and Methods."

Gene Expression and Protein Profiling of NTR and Trx h in Germinating Embryos

Expression of HvTrxh1, HvTrxh2, HvNTR1, and HvNTR2 genes was analyzed by semiquantitative reverse transcription (RT)-PCR using total RNA from embryos dissected from mature seeds and seeds at different time points during germination. The appearance of the corresponding proteins was monitored by western-blot analyses of soluble proteins extracted from the embryo during germination. Transcripts of both HvTrxh1 and HvTrxh2 were present in embryos from mature seeds and remained constant during germination (Fig. 2A ). Trx h protein, however, increased slightly in amount from 24 h after imbibition (Fig. 2B), corresponding to the time of radicle protrusion, and then remained constant up to 144 h. HvNTR1 and HvNTR2 transcripts were both detected at low levels in embryos from mature seeds (Fig. 2A). Expression increased considerably up to 72 h after imbibition, then started to decrease. NTR protein (Fig. 2B) was detected at low levels in embryos 4 h after imbibition, increased up to 48 h, and then remained essentially constant. For both Trx h and NTR, the western-blotting profiles represent the combined appearance of all isoforms.

View larger version (56K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Gene expression and protein appearance for Trx h and NTR isoforms in barley embryos during germination (0–144 h). A, RT-PCR analysis of HvTrxh1, HvTrxh2, HvNTR1, and HvNTR2. One representative gel is shown from three independent replicates. Relative band intensities were normalized to the 18S rRNA band intensity (100%). Each histogram represents the mean ± SD obtained from three independent RT-PCR reactions. B, Western blot of embryo protein extracts using antibodies against Trx h and NTR.

The cereal seed aleurone layer has a key role in germination, responding to hormone signals from the embryo and producing storage reserve mobilizing enzymes. Isolated aleurone layers can be maintained in buffer, providing a well-defined system for analysis of hormonal regulation of seed germination (Fath et al., 2001). Aleurone layers were incubated in buffer containing GA or ABA and harvested at time points between 6 and 48 h. HvTrxh1 and HvTrxh2 expression was detected in aleurone layers incubated with or without the addition of GA or ABA and the expression level varied only slightly (Fig. 3A ). Trx h protein, however, was hardly detected in aleurone layers after 6-h incubation (Fig. 3B) and the level increased slightly during the course of the incubation. A similar increase was observed in aleurone layers treated with GA or ABA, suggesting that these hormones do not regulate gene expression or protein accumulation of HvTrxh1 and HvTrxh2.

View larger version (62K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Gene expression and protein appearance for Trx h and NTR isoforms in aleurone layers responding to GA, ABA, and buffer without hormones (control) at various time points (6–48 h). A, RT-PCR analysis of HvTrxh1, HvTrxh2, HvNTR1, and HvNTR2. One representative gel is shown from three independent replicates. Relative band intensities were normalized to18S rRNA band intensity (100%). Each data point represents the mean ± SD obtained from three independent RT-PCR reactions. B, Western blot of aleurone protein extracts using antibodies against Trx h and NTR. C, Coomassie- and silver-stained 2-D gels and 2-D western blotting using Trx h antibody in aleurone layers treated in the presence and absence of GA and ABA. The Trx h spot is marked by an arrow.

2-D Gel Electrophoresis Pattern of Trx h in Aleurone Layers

HvTrxh1 and HvTrxh2 were identified in two and one 2-D gel spots, respectively, in mature barley embryos (Maeda et al., 2003). One HvTrxh1 spot and the HvTrxh2 spot decreased at radicle elongation (Bønsager et al., 2007). In aleurone layers dissected from mature seeds, HvTrxh2 was absent and HvTrxh1 appeared in one spot (Maeda et al., 2003). Trx h transcript levels in aleurone layers appear to be independent of GA and ABA. Therefore, 2-D western blotting was performed to examine whether the individual protein isoforms were affected by hormone treatment. A single spot with approximate 12.5-kD molecular mass and pI 5.0 was detected in all blots and on 2-D gels stained with Coomassie Blue and silver nitrate (Fig. 3C). The spot was excised from the Coomassie-stained gel for in-gel digestion with trypsin and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Five peptide masses matched HvTrxh1 (AAP72290), giving 20% sequence coverage. The abundance of the spot was similar in aleurone layers treated with or without GA or ABA, in agreement with gene expression and the 1-D western blot, indicating that synthesis of the protein was not regulated by hormones. The HvTrxh1 spot was hardly detected in aleurone layers incubated for 12 or 18 h (Fig. 3C) and increased in intensity during incubation, in agreement with the 1-D western blot. Identification of only the HvTrxh1 protein in aleurone layers is in agreement with previous findings (Maeda et al., 2003), but is in contrast to detection of transcripts corresponding to both Trx h genes.

Expression, Purification, and Biochemical Characterization of HvNTR1 and HvNTR2

To confirm that the cDNA sequences encoded active NTR and to compare the kinetic properties of the two isoforms, recombinant proteins carrying an amino-terminal His₆-tag were produced in Escherichia coli. HvNTR1 and HvNTR2 were found in the soluble fraction of the E. coli transformant culture after induction with isopropyl-β-D-thiogalactopyranoside (IPTG) and were recognized both by an antibody recognizing the His-tag and an antibody raised against wheat NTR (data not shown). SDS-PAGE of cell extracts showed a prominent polypeptide band of the expected molecular mass (Fig. 4A , lanes 2 and 5), and the recombinant His₆-HvNTR1 and His₆-HvNTR2 were purified from the crude extracts by nickel affinity chromatography (Fig. 4A, lanes 3 and 6) in yields of 30 and 10 mg/L, respectively. Proteins were yellow with absorption maxima at 270, 378, and 454 nm (Fig. 4B) typical for flavoproteins (Jacquot et al., 1994; Serrato et al., 2002).

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Expression, purification, and chemical properties of NTR. A, SDS-PAGE of NTR overexpression in E. coli, purification, and thrombin cleavage. Total protein from E. coli harboring pET15b-HvNTR1 (lanes 1 and 2) and pET15b-HvNTR2 (lanes 4 and 5), respectively, before (lanes 1 and 4) and 3.5 h after (lanes 2 and 5) induction with IPTG; purified His-HvNTR1 and His-HvNTR2, respectively (lanes 3 and 6); and HvNTR2 after thrombin cleavage to remove the His-tag (lane 7). B, Absorption spectrum of recombinant HvNTR2. C, MALDI MS spectrum of intact His-HvNTR2 and after cleavage with thrombin.

Enzyme Activity

Kinetic parameters for activity of HvNTR1 and HvNTR2 were determined using barley HvTrxh1 and HvTrxh2 and, for comparison, also with E. coli Trx as substrate (Table I ). K_m values indicate that HvNTR1 and HvNTR2 have similar affinities for HvTrxh1 and HvTrxh2. Activity of HvNTR1 with HvTrxh1, represented by k_cat, was almost twice that with HvTrxh2. In contrast, the k_cat of HvNTR2 was the same for HvTrxh1 and HvTrxh2. The k_cat of HvNTR2 with E. coli Trx was similar to that of barley HvNTR1 with barley Trx h isoforms, whereas the K_m for E. coli Trx was very high. The catalytic efficiency (k_cat/K_m) for E. coli Trx was thus 100-fold lower than for HvTrxh1 and HvTrxh2, indicating that a noncognate Trx is not a good substrate for the NTR. Similarly, thioredoxin reductases from Caenorhabditis elegans (Lacey and Hondal, 2006) and Plasmodium falciparum (Krnajski et al., 2001) also show high activity toward E. coli Trx, but with a high K_m. Kinetic parameters were also determined using Arabidopsis NTR (AtNTR; Jacquot et al., 1994), which had lower affinity and lower activity for barley Trx h isoforms compared with barley NTRs (Table I). Conversely, both AtNTR and E. coli NTR showed high affinity for their own Trxs, K_m being 1.1 (Jacquot et al., 1994) and 1.9 to 2.4 µM (Miranda-Vizuete et al., 1997), respectively, similar to the K_m values determined here for the barley NTR/Trx h pairs. These data underline that NTR from different species prefer their cognate Trx h as substrate.

View larger version (16K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Time course of HvTrxh1 and HvTrxh2 reduction by HvNTR2 as monitored by reduction of DTNB at various pH values (5, 5.7, 6.5, and 7.4). Control shows the time course of DTNB reduction by HvNTR2 without addition of Trx h.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

Despite the different tasks proposed for the NTR/Trx system during germination (Wong et al., 2002, 2004), most experimental evidence was obtained at more basic pH values than expected to be present in vivo. Under weak acidic conditions in the starchy endosperm during germination, the Trx N-terminal active-site Cys (e.g. C46_HvTrxh2), acting as a nucleophile in catalysis of the disulfide exchange reaction (Kallis and Holmgren, 1980; Maeda et al., 2006) and with pK_a of 6.3 to 7.5 (in E. coli Trx; Setterdahl et al., 2003), may be protonated and hence inactive. Therefore, the central question of whether the NTR/Trx system is active at the pH of the endosperm during germination was not clearly addressed. This work demonstrates, however, that recombinant HvNTR1 and HvNTR2 are able to reduce Trx h at pH 5.0 with essentially retained affinity, but slightly decreased activity, which supports that the NTR/Trx system operates in germination.

Functional analysis was complemented by gene expression and protein profiling during germination because, for NTR/Trx pairs to interact, they must be present in the same tissue at the same time. Profiling experiments also provide information about gene expression, posttranscriptional regulation, and posttranslational regulation. Transcripts corresponding to both Trx h and NTR isoforms were detected in embryos and aleurone layers at all time points, suggesting that the NTR/Trx system is active in these tissues during germination. Levels of Trx h transcripts were relatively constant, whereas NTR transcript accumulation showed greater modulation, as characteristic for genes encoding important regulatory proteins. The comparison of Trx h gene expression and protein profiles illustrates the general problem with predicting protein expression levels from mRNA data. Differences can be due to posttranscriptional modification controlling the protein translation rate (Day and Tuite, 1998), protein modification or degradation, and protein transport to or from cytoplasm (Gygi et al., 1999; You and Yin, 2000). Distinct transcript accumulation patterns were observed for HvNTR1 and HvNTR2, indicating that the isoforms are differentially regulated and may have individual functions in seed germination. HvNTR2 perhaps has a specific role in germination because its expression in the aleurone layer was affected by GA. HvNTR1 expression level is much lower in aleurone than embryo and the NTR protein profile in the aleurone layer closely resembles the HvNTR2 gene expression pattern. In combination, gene expression and protein profiling suggest that, whereas both NTR and Trx h isoforms are present in the embryo, with HvTrxh1 protein more prominent during germination, HvNTR2 and HvTrxh1 may be the dominant isoforms in the aleurone layer during germination. Isolation of the promoter regions for the two genes would be one way to further study the regulation.

The observed increase of NTR gene expression in germinating embryos would be expected to lead to an increase in the proportion of reduced Trx h and hence reduced Trx h target proteins. These results agree with protein disulfides becoming more reduced during germination (Marx et al., 2003; Alkhalfioui et al., 2007b). The reported elevated activity during germination of oxidative pentose phosphate pathway enzymes (Lozano et al., 1996) could provide the NADPH required for activation of the NTR/Trx system in embryos. In contrast, the decrease of HvNTR2 expression in aleurone layers in response to GA at early incubation time points suggests an initial low level of reduced Trx h and target proteins in the aleurone layer. Several potential Trx target proteins (Yamazaki et al., 2004; Gelhaye et al., 2006) are involved in protection against reactive oxygen species, generated as by-products of lipid metabolism in the aleurone layer (Bethke et al., 2002) and implicated in the initiation of the cell death program (Fath et al., 2001). Down-regulation of HvNTR2 expression by GA is predicted to decrease the proportion of reduced Trx h, resulting in modulation of target protein activity. Therefore, regulation of the NTR/Trx system by hormones could affect cell death signaling in aleurone layer cells.

It still remains to be determined whether NTR isoforms are localized in the same cell compartments. In Arabidopsis, both mitochondrial and cytosolic forms (Reichheld et al., 2005) of NTR have been identified.

	CONCLUSION

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 
This study demonstrates time interactions between two NTR and two Trx h isoforms from the same organism. The results support a functional role of the NTR/Trx system during germination and suggest that the members of the barley seed NTR/Trx system can function interchangeably. Future proteomics-based studies of individual NTR and Trx isoforms in conjunction with determination of intracellular localization and promoter structure may clarify their differential expression pattern in response to hormones and their possibly divergent in vivo functions. Finally, heterologous expression of both NTR and Trx h isoforms provides a basis for design and characterization of mutants to investigate the interaction between NTR and Trx h at the level of molecular structures.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION CONCLUSION MATERIALS AND METHODS LITERATURE CITED

 

Plant Material

Embryos from germinated seeds were prepared from the malting barley (Hordeum vulgare ‘Barke’) provided by Sejet Plantbreeding. Mature seeds were sterilized in 5% sodium hypochlorite for 30 min and rinsed several times with water. Seeds were germinated for 0, 4, 24, 48, 72, 96, 120, and 144 h, frozen, and stored as described (Bønsager et al., 2007). Embryos were dissected from seeds prior to protein and RNA extraction.

Aleurone layers were prepared from embryoless half-grains (Hynek et al., 2006) of barley ‘Himalaya’ (purchased from Washington State University). Isolated aleurone layers (100 mg fresh weight) were incubated in 2 mL of 20 mM sodium succinate (pH 4.2), containing 20 mM CaCl₂, 50 µg/mL ampicillin, and 5 µg/mL nystatin. Where appropriate, GA or ABA (5 µM) was added. Incubation was performed at room temperature with continuous gentle shaking. Aleurone layers were harvested at various time points (6–48 h), washed four times with 2 mL of the incubation medium without addition of antibiotics or hormones, frozen in liquid nitrogen, and stored at –80°C until use.

RT-PCR Analysis

Total RNA was extracted from tissues using the RNeasy plant mini kit (Qiagen) and treated with RNase-Free DNase (Qiagen). RT-PCR was performed using a PTC-200 DNA Engine Peltier thermal cycler (Bio-Rad) and a one-step RT-PCR kit (Qiagen) according to the manufacturer's recommendations. Barley 18S rRNA showing invariant expression across the samples was amplified in parallel. The optimal number of amplification cycles (between 15 and 45) for each set of primers was determined at the exponential phase range of amplification. To control for possible genomic DNA contamination, parallel reactions were carried out where reverse transcriptase activity was inactivated by incubation at 95°C. A negative control lacking template RNA was included for each set of RT-PCR reactions. Reactions were performed in triplicate. Amplification products were separated by agarose gel electrophoresis and quantified using ImageJ software (W.S. Rasband; 1997–2007; National Institutes of Health; http://rsb.info.nih.gov/ij). Signal intensities were normalized with respect to 18S rRNA from the same sample. Primers used were trxh8 and trxh9 for HvTrxh1, trxh10F and trxh10R for HvTrxh2, ntr3F and ntr3R for HvNTR1, and ntr4F and ntr4R for HvNTR2 (Supplemental Table S1).

Sequence Analysis

Multiple alignment of the region marked in Supplemental Figure S1 was performed using ClustalW (http://www.ebi.ac.uk/Tools/clustalw). A phylogenetic tree was constructed using the GeneBee TreeTop phylogenetic tree prediction server (http://www.genebee.msu.su/genebee.html). National Center for Biotechnology Information (NCBI) accessions used for the analysis were: Os, rice (Oryza sativa), Os1 (NP_001047911), Os2 (EAY87270), Os3 (EAZ24372), Os4 (BAD33510), Os5 (NP_001057531), Os6 (EAZ00754), Os7 (EAZ36842), Os8 (NP_001060515); At, Arabidopsis (Arabidopsis thaliana), At1 (Q39242), At2 (NP_195271), At3 (1VDC), At4 (AAO42318), At5 (AAO42318), At6 (CAA80656), At7 (AAL08250), At8 (NP_565954); Hv, H. vulgare, Hv1 (ABY27300), Hv2 (ABX09990), Hv3 (TC132362); Ta, wheat (Triticum aestivum; CAD19162); Mt, Medicago truncatula, Mt1 (ABH10138), Mt2 (ABH10139); Ol, Ostreococcus lucimarinus (XP_001422184); Te, Thermosynechococcus elongates (NP_682714); Ns, Nostoc sp. (NP_484780); Pm, Prochlorococcus marinus (NP_893267); Nc, Neurospora crassa (P51978); Pc, Penicillium chrysogenum (P43496); Sc, Saccharomyces cerevisiae (P29509); Ec, Escherichia coli (P09625); Yp, Yersinia pestis (NP_404967); and Hi, Haemophilus influenzae (P43788).

Protein Extraction and Western-Blot Analysis

Frozen tissues were dried under vacuum and ground to a fine powder using a mortar and pestle. Soluble proteins were extracted in 5 mM Tris-HCl, 1 mM CaCl₂ (pH 7.5) with the protease inhibitor cocktail Complete (Roche; Finnie and Svensson, 2003). Protein concentration was determined using the Popov assay with bovine serum albumin as standard (Popov et al., 1975).

Proteins (1.5 µg total protein from the aleurone layer or 6 µg total protein from the embryo) were separated on 4% to 12% Bis-Tris NuPAGE gels (Invitrogen) and stained with colloidal Coomassie Brilliant Blue G-250 (Candiano et al., 2004). Proteins were electroblotted onto a nitrocellulose membrane (Hybond-N; GE Healthcare) and probed using rabbit anti-wheat Trx h or rabbit anti-wheat NTR antibodies. Secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase (Dako Cytomation) were detected by enhanced chemiluminescence (Thorpe and Kricka, 1986) or the nitroblue tetrazolium/bromochloro indolyl phosphate colorimetric method, respectively.

2-D Gel Electrophoresis and Western Blotting

Protein extracts from 100-mg aleurone layers were desalted on NAP-5 columns (GE Healthcare). Aliquots containing 50 µg of protein were precipitated by ammonium acetate (0.1 M) in methanol (Vensel et al., 2005) and redissolved in reswelling buffer (7 M urea, 2 M thiourea, 2% [v/v] CHAPS, 0.5% [v/v] immobilized pH gradient [IPG] ampholytes 3–10 [Amersham Biosciences], 1.2% [v/v] Destreak reagent [hydroxyethyl disulfide], and a trace of bromphenol blue). First-dimension isoelectric focusing was done using 18-cm linear IPG strips, pI 3 to 10, and an IPG phor (GE Healthcare). IPG strips were subsequently equilibrated as described (Finnie et al., 2002) for second-dimension electrophoresis on Excel SDS XL (18 x 24 cm) 12% to 14% gradient gel (GE Healthcare) using Multiphor II (GE Healthcare) according to the manufacturer's recommendations and stained with either silver nitrate (Heukeshoven and Dernick, 1985) or colloidal Coomassie Brilliant Blue G-250 (Candiano et al., 2004). For electroblotting onto nitrocellulose, gels were first equilibrated for 30 min in NuPAGE transfer buffer (Invitrogen) containing 15% methanol.

In-Gel Digestion and Protein Identification

Spots cut out from Coomassie-stained gels were in-gel digested with trypsin (Promega) as described (Finnie et al., 2002). Peptides were micropurified (Gobom et al., 1999) and eluted directly onto the MALDI target in 5 µg/µL -cyano-hydroxy-cinnamic acid in 70% acetonitrile and 0.1% trifluoroacetic acid. An Ultraflex II MALDI-TOF-TOF mass spectrometer (Bruker-Daltonics) was used in positive ion reflector mode and spectra were analyzed using FlexAnalysis software (Bruker-Daltonics). Spectra were calibrated externally using a tryptic digest of β-lactoglobulin. Internal calibration was carried out with trypsin autolysis products (m/z 842.51 and m/z 2211.10). Peptide mass data were searched against the NCBI nonredundant database using Biotools (Bruker Daltonics) software and Mascot (MatrixScience) with the following parameters: monoisotopic mass tolerance, 80 ppm; allowed missed cleavages, 1; allowed modifications, carbamidomethylation of Cys (global) and oxidation of Met (partial).

Isolation and Cloning of HvNTR1 and HvNTR2

Total RNA was extracted from embryos and contaminating genomic DNA was removed as described above. Conserved regions from plant genes encoding NTR were used to design primers (ntr1F and ntr1R). RT-PCR was performed using the one-step RT-PCR kit (Qiagen) according to the manufacturer's instructions. The amplified fragment (700 bp) was cloned in the pDrive cloning vector (Qiagen). Sequencing of cloned fragments (MWG-Biotech AG) distinguished two types of internal NTR sequences, cDNA1 and cDNA2. Because cDNA1 was 98% identical to TC142091 and cDNA2 was 100% identical to TC141301, these TC sequences were used to design gene-specific primers for RT-PCR amplification from embryo RNA and sequencing of the 5' end (primers ntr4F and ntr4R) and 3' end (primers ntr5F and ntr5R) of the cDNA2 coding sequence and the 3' end of the cDNA1 coding sequence. Because TC 142091 did not include a complete coding sequence and lacked the 5' untranslated region, the untranslated region from wheat NTR (accession no. AJ421947, 97% identical to cDNA1) was used to design primers (ntr2F and ntr2R) for isolation of the 5' end of the cDNA1 coding sequence. Amplicons were cloned in pDrive cloning vectors and sequenced. Finally, cDNAs containing the entire coding regions of HvNTR1 and HvNTR2 were amplified by RT-PCR from embryo RNA using primers ntr2F and ntr6R and ntr4F and ntr5R, respectively, and cloned to give pDrive-HvNTR1 and pDrive-HvNTR2. Primer sequences are listed in Supplemental Table S1.

Expression, Purification, and Chemical Properties of Recombinant HvNTR1 and HvNTR2

The restriction sites NdeI and BamHI were introduced at the ends of the HvNTR1 coding sequence by primers ntr10F and ntr10R, using HotStar HiFidelity PCR (Qiagen) and pDrive-HvNTR1 as template. pDrive-HvNTR2 was used as template with primers ntr8F and ntr8R to introduce NdeI and XhoI sites. After cleavage by the appropriate restriction enzymes, the inserts were ligated into pET15b (Novagen) to give pET15b-HvNTR1 and pET15b-HvNTR2. Sequences were verified and constructs were used to transform E. coli strain Rosetta (DE3). Cells were grown at 37°C in Luria-Bertani medium supplemented with 100 µg/mL ampicillin and 5 µg/mL chloramphenicol to an OD₆₀₀ of 0.6. Cultures were induced by 100 µM IPTG for 3.5 h. Cells were harvested by centrifugation and frozen at –20°C until use.

Frozen pellets were resuspended in Bugbuster protein extraction reagent including Benzonase (Novagen) and shaken for 20 min at room temperature. After centrifugation (14,000g for 20 min, 4°C), recombinant protein in the supernatant was purified by a His-Trap HP column (Amersham Biosciences) as described (Maeda et al., 2006) and eluted fractions were analyzed by SDS-PAGE. Purified proteins were desalted on PD-10 columns (GE Healthcare) in 10 mM Tris-HCl (pH 8.0).

Protein concentration was determined by amino acid analysis. The absorption spectrum was recorded for 8.7 µM NTR in 10 mM Tris-HCl (pH 8.0) at room temperature. For cleavage of the N-terminal His-tag, purified His₆-tag HvNTR2 (0.1 µg/µL) was treated with immobilized thrombin (Calbiochem) at 1:100 (w/w) thrombin:fusion in 0.6 mM NaCl, 50 mM Tris-HCl (pH 6.0) for 24 h at 24°C. Mass spectrometric analysis of intact proteins was performed using 20 pmol His-HvNTR2 or cleaved HvNTR2 after micropurification of samples as described above, but using Poros 20 R1 (Applied Biosystem) as column material and eluting in sinapinic acid (10 mg/mL) in 70% acetonitrile and 0.1% trifluoroacetic acid. External calibration of spectra was performed using protein standard II (Bruker-Daltonics).

Enzyme Activity Assay

Activity of NTR was measured at 25°C using DTNB as the final disulfide substrate as described (Miranda-Vizuete et al., 1997) with slight modifications. The reaction mixture contained 100 mM potassium phosphate (pH 5.0–7.4), 10 mM EDTA, 200 µM DTNB (Sigma-Aldrich), 200 µM NADPH (Sigma-Aldrich), and Trx (1–10 µM) from barley or E. coli (Promega). The reaction was started by the addition of NTR (40 nM) and the rate of reduction of DTNB was calculated from the A₄₁₂. As 1 mol Trx-(SH)₂ reduces 1 mol DTNB to yield 2 mol TNB (2-nitro-5-thiobenzoic acid) with a molar extinction coefficient of 13,600 M^–1 cm^–1 (Ellman, 1959), a molar extinction coefficient of 27,200 M^–1 cm^–1 was applied for quantification. The extinction coefficient of TNB varied by 1% to 4% from this value in the pH range 5.0 to 7.4 employed in this work (data not shown).

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers EU314717, EU250021, ABY27300, and ABX09990.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Multiple alignment of primary sequences of NTRs from different sources using ClustalW.

Supplemental Table S1. List of forward (F) and reverse (R) primers.

	ACKNOWLEDGMENTS

 
We thank B. Buchanan (University of California, Berkeley) for rabbit anti-wheat Trx h, F.J. Cejudo (University of Seville, Spain) for rabbit anti-wheat NTR, and J.P. Jacquot (INRA, Nancy, France) for recombinant Arabidopsis NTR. We also wish to thank P. Hägglund and K. Maeda for helpful discussions, B.C. Bønsager for assistance with embryo samples, and K. Rasmussen, A. Blicher, and B. Andersen for technical assistance.

Received November 21, 2007; accepted December 12, 2007; published December 27, 2007.

	FOOTNOTES

 
¹ This work was supported by the Iranian Ministry of Science, Research and Technology, the Danish Agriculture and Veterinary Research Council, and the Danish Centre for Advanced Food Studies.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Christine Finnie (csf{at}biocentrum.dtu.dk).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.113639

^* Corresponding author; e-mail csf{at}biocentrum.dtu.dk.

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