Introduction

Reactive oxygen species (ROS), including superoxide radicals, hydroxyl radicals, and hydrogen peroxide, are generated during the incomplete reduction of molecular oxygen to water. In addition, biotic and abiotic stresses can lead to the alteration of cellular redox homeostasis, resulting in oxidative stress (Rodriguez Milla et al. 2003). High levels of ROS can damage biological molecules such as nucleic acids, lipids, and proteins. Therefore, organisms have developed several nonenzymatic and enzymatic systems to protect against oxidative damage caused by ROS. The major nonenzymatic antioxidant factors are carotenoids, tocopherols, reduced glutathione (GSH), and ascorbate. Enzymatic systems include glutathione peroxidases (GPXs), peroxiredoxins, superoxide dismutases, catalases, and ascorbate peroxidases (Navrot et al. 2006). Among these antioxidant enzymatic systems, GPXs play important roles in scavenging ROS and protecting cells from oxidative damage (Navrot et al. 2006; Iqbal et al. 2006). As an important ROS scavenger, GPXs have broad substrate specificities and a high affinity for H2O2 (Brigelius-Flohe and Flohe 2003). Their principal activity was thought to be catalyzing the reduction of H2O2 and lipid hydroperoxides to water and alcohol, respectively, using glutathione as the electron donor (Chang et al. 2009; Fu et al. 2002).

In plants, GPX proteins can reduce peroxides such as H2O2, cumene hydroperoxide (Cum-OOH), and tert-butyl hydroperoxide (t-BuOOH) using thioredoxin (Trx) as the electron donor (Jung et al. 2002; Iqbal et al. 2006; Wang et al. 2007). A recent study showed that the knockout of the Arabidopsis AtGPX8 gene led to increased sensitivity to oxidative damage during root elongation (Gaber et al. 2012). Conversely, overexpression of AtGPX8 in transgenic Arabidopsis resulted in reduced sensitivity to oxidative damage (Gaber et al. 2012). Arabidopsis AtGPX3 mutants exhibited enhanced sensitivity to H2O2 treatment during seed germination and seedling development and enhanced H2O2 production in guard cells (Miao et al. 2006). Reduced expression of Arabidopsis chloroplast GPX genes (AtGPX1 and AtGPX7) led to reduced tolerance to photooxidative stress (Chang et al. 2009). In addition to their role in oxidative stress, plant GPXs also play roles in signaling pathways. For example, Arabidopsis GPX3 (AtGPX3) acts as an oxidative signal transducer in abscisic acid and drought stress signaling (Miao et al. 2006). Therefore, plant GPX genes have extensive functional divergence.

Plant GPXs form gene families, which comprise eight and six members, the Arabidopsis and poplar genomes, respectively (Gaber et al. 2012; Navrot et al. 2006). To date, studies on GPXs in plants have focused mainly on angiosperms such as Arabidopsis, poplar, tomato, and rice (Rodriguez Milla et al. 2003; Navrot et al. 2006; Herbette et al. 2002; Li et al. 2000; Agrawal et al. 2002). In contrast, there is little information describing the molecular characteristics of GPXs in gymnosperms. Gymnosperms such as cycads, ginkgo, gnetales, and conifers represent a major group of extant seed plants (Burleigh et al. 2012). Conifers, which are usually perennial woody plants that are widespread in mountainous areas (Zeng et al. 2005), are by far the most abundant gymnosperms (Stefanoviac et al. 1998). During their long life cycle, conifers have to withstand multiple severe stresses including heat and cold shock, wounding, and drought stress (Zeng et al. 2005). All these stressors induce increased production of ROS. As important ROS scavengers, GPXs help maintain oxidative homeostasis, which support the survival of conifers in extreme environments.

In this study, we cloned four GPX genes from the gymnosperm Pinus tabulaeformis, which is a conifer that is widespread across northern and central China. We then conducted a systematic characterization of P. tabulaeformis GPXs including gene expression patterns and biochemical and structural characteristics. This study provides novel insights into the functional divergence and catalytic properties of the GPX family in gymnosperms.

Materials and Methods

Molecular Cloning

The GenBank Pinus taeda EST database was searched using the tBLASTN program (http://www.ncbi.nlm.nih.gov/genomes/PLANTS/PlantList_or.html#EST) with default parameters. Arabidopsis GPX protein sequences were used as query sequence. The accession numbers of ESTs and their e value are listed in Supplemental Table S1. Four genes encoding putative full length GPX proteins were identified. Four pairs of specific primers were designed based on the P. taeda GPX genes to amplify the P. tabulaeformis GPX cDNAs. Fresh leaves of P. tabulaeformis were used for total RNA isolation using a BioTeKe Total RNA kit (BioTeKe Corp., Beijing, China). Total RNAs were treated with RNase-free DNase I (Promega, Madison, WI, USA). First-strand cDNA was synthesized using a TaKaRa RNA PCR kit (AMV) Ver. 3.0 (Takara, Shiga, Japan). The polymerase chain reaction (PCR) was performed in a volume of 25 μl containing 1 μl of first-strand cDNA, 5 μl of TaKaRa 5× PCR buffer, 0.12 μl TaKaRa pyrobest polymerase, and 0.5 pmol of each primer. PCR conditions were optimized to initial denaturation of 3 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 40 s at 55 °C, and 60 s at 72 °C, followed by a final extension of 5 min at 72 °C. PCR products recovered from a 1 % agarose gel were cloned into the pGEM-T vector (Promega, Madison, WI, USA) and sequenced in both directions. The four genes from P. tabulaeformis were named PtaGPX1, PtaGPX2, PtaGPX3, and PtaGPX4, respectively.

Expression of GPX Genes in P. tabulaeformis Tissues

To investigate the expression pattern of GPX genes in P. tabulaeformis tissues, total RNAs were isolated from root, phloem in the root, stem, phloem in the stem, leaf, and bud using a BioTeKe Total RNA kit (BioTeKe Corp., Beijing, China). Total RNAs were treated by DNase I and reverse transcribed into cDNA. PCR conditions were optimized to initial denaturation of 3 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 40 s at 60 °C, and 60 s at 72 °C, followed by a final extension of 5 min at 72 °C. In all PCR analyses, the Actin gene was used as an internal control. PCR products from each sample were analyzed using a 1 % agarose gel and were validated by DNA sequencing. Independent biological triplicates were used in all gene expression analyses.

Homology Modeling

The crystal structure of the reduced form of poplar glutathione peroxidase (PtGPX5) (Protein Data Bank [PDB] code: 2p5q) was used as the template for constructing structural models of Pinus GPX. The oxidized form of Pinus GPX protein was modeled based on the X-ray structure of oxidized poplar PtGPX5 (PDB code: 2p5r). Sequences were aligned using the Align 2D structural alignment program (Homology Module, InsightII), and structures were then built using the Modeler module of InsightII. The Profile-3D program in InsightII was used to verify all structures. The structural models were selected according to the model evaluation score calculated by Profile-3D and verified using Ramachandran plots (Laskowski et al. 1993).

Protein Expression and Purification

The open reading frame (including the stop codon) encoding the four P. tabulaeformis GPX proteins were subcloned into the modified ΔpET30a plasmid expression vector (Yang et al. 2009). The resulting plasmids were used to transform Escherichia coli tuner (DE3). Colonies containing the appropriate insert were identified by sequencing. The Cys44, Cys73, Cys92, and Glu80 residues of PtaGPX3 were individually replaced with alanine. DNA sequencing confirmed that all of the desired mutants were successfully generated. All mutant cDNAs were subcloned into the ΔpET30a plasmid to generate expression constructs. An overnight culture of E. coli tuner containing the recombinant ΔpET30a plasmids was diluted 1:100 and grown until the optical density (A 600) reached 0.5. Isopropyl beta-d-thiogalactoside (IPTG) was added to the culture at a final concentration of 0.05 mM, and the incubation was continued overnight at 37 or 20 °C. Bacteria were harvested by centrifugation at 10,000×g for 3 min at 4 °C, resuspended in binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4), and disrupted by cold sonication. The particulate material was removed by centrifugation at 10,000×g for 10 min at 4 °C, and the supernatant was loaded onto a Ni Sepharose High Performance column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) that had been pre-equilibrated with binding buffer. The target protein that bound to the Ni Sepharose High Performance column was eluted with elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 0.5 M imidazole, pH 7.4).

Enzyme Assays and Kinetic Studies

The peroxidase activity was measured by the decrease in the A 340 in a reaction mixture containing 150 mmol/L Tris–HCl, pH 8.0, 7.5 mmol/L EDTA, 1 mmol/L H2O2, various amounts of protein, and an electron donor, either the Trx system consisting of 0.2 mM NADPH, 9 μmol/L E. coli Trx, 0.5 μmol/L E. coli Trx reductase or the GSH system containing 0.2 mmol/L NADPH, 2 mmol/L GSH, and 1.5 U glutathione reductase. For the investigation of substrate specificity, H2O2 was replaced with various concentrations of t-butyl hydroperoxide or cumene hydroperoxide. Data represent the mean of at least three independent experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on a 14 % separating gel and a 5 % stacking gel. Protein concentrations were determined by measuring absorbance at 280 nm. Steady-state kinetics of the enzyme were studied in assays with various concentrations of E. coli Trx and H2O2. The apparent Km value of Trx was determined using a Trx range from 0.5 to 25 μM at a fixed H2O2 concentration of 1.0 mM. The apparent Km value for H2O2 was determined using an H2O2 range from 0.005 to 1.0 mM at a fixed Trx concentration of 5 μmol/L. The kinetic parameters were derived using nonlinear regression as implemented in the Hyper32 program available from http://homepage.ntlworld.com/john.easterby/hyper32.html.

Results

Sequence Characterization of the Pinus GPX Genes

Four DNA fragments encoding putative P. tabulaeformis GPXs were amplified using reverse transcription (RT)-PCR and were named PtaGPX1, PtaGPX2, PtaGPX3, and PtaGPX4 (GenBank accession numbers KF032617, KF032618, KF032619, and KF032620, respectively). PtaGPX1, PtaGPX2, and PtaGPX3 encoded proteins of 170 amino acids with molecular weights of 18.75, 18.64, and 18.95 kDa, respectively. PtaGPX4 encoded a protein of 173 amino acids and a predicted molecular weight of 19.30 kDa. Based on the National Center for Biotechnology Information conserved domain analysis, all four proteins contained GPX domains, suggesting that these genes belonged to the GPX gene family.

The PtaGPX protein sequences were compared with ten GPXs from other plant species (Fig. 1). The protein sequence identities among the four Pinus GPXs ranged from 63.0 to 88.8 %. The protein sequence identities between the four Pinus GPXs and other ten plant GPX proteins ranged from 47.6 to 70.3 %. The high sequence identity between the PtaGPXs and other plant GPXs further confirmed that the four PtaGPX genes cloned in this study belonged to GPX gene family.

Fig. 1
figure 1

Sequence alignment of plant GPX proteins. Residues that were conserved in all plant GPXs are shaded. Black triangles show the conserved Cys and Glu residues in alignment positions 113, 142, 149, and 161

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Expression Patterns of the GPX Genes in P. tabulaeformis

To investigate the expression pattern of Pinus GPX genes in different tissues, RT-PCR was performed on the total RNA isolated from the roots, phloem in roots, stems, phloem in stems, leaves, and buds. Data revealed that the four Pinus GPX genes were expressed in all tissues examined (Fig. 2), suggesting that their transcripts are distributed widely in P. tabulaeformis.

Fig. 2
figure 2

Expression of the GPX genes in various tissues of P. tabulaeformis. Lanes 1–6 roots, phloem in the roots, stems, phloem in the stems, leaves, and buds, respectively. Actin was used as the internal standard

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Structural Modeling

To investigate the three-dimensional (3D) structure of the four Pinus GPX proteins, we conducted structure modeling based on the X-ray structure of the reduced form of the poplar glutathione peroxidase (PtGPX5). Profile-3D analysis showed that, aside from Tyr51 of PtaGPX2 and 3, all other residues of PtaGPX1, 2, 3, and 4 were scored positive (Supplementary Figure S1). Ramachandran plots showed that more than 93 % of the amino acids of all of the models were located in the most favored regions (Supplementary Figure S2). Profile-3D analysis and Ramachandran plots suggested that the predicted 3D structures were folded reliably.

Structural modeling revealed that the conformation of structural elements, including α-helices and β-strands, were similar in the four Pinus GPX proteins and the X-ray structure of PtGPX5. The 3D structures of Pinus GPXs consisted of four α-helices and six β-strands (Fig. 3). Among six β strands, four β-strands (β3 − 6) formed a twisted central β-sheet, whereas the other two strands (β1 and 2) folded into a β-hairpin. Three α-helices (α1, 2, and 4) were located on one side of the twisted central β sheet, and helix 3 was on the opposite side. Structural modifications between proteins were present in one loop region, which may indicate functional diversity between the four Pinus GPX proteins.

Fig. 3
figure 3

Structural analysis of Pinus GPX proteins. a Structural superposition of four Pinus GPX proteins. b, c Putative three-dimensional structures of PtaGPX3 under reducing and oxidizing conditions, respectively

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Biochemical Characterization of the Pinus GPX Proteins

To assess the enzymatic activities of the four Pinus GPX proteins, they were overexpressed in E. coli as mainly soluble proteins after induction with IPTG at 37 °C. The four soluble GPX proteins were purified using a Ni Sepharose High Performance column (Fig. 4). Previous studies showed that some GPX proteins could reduce H2O2, cumene hydroperoxide (Cum-OOH), and tert-butyl hydroperoxide (t-BuOOH) using GSH and thioredoxin (Trx) as reducing agents (Navrot et al. 2006; Jung et al. 2002). Therefore in this study, we also used GSH and Trx as reducing agents to investigate the activities of the Pinus GPX proteins.

Fig. 4
figure 4

SDS-PAGE of purified GPX proteins. Lane 1 molecular mass markers with the sizes shown on the left in kDa; lanes 2–9 PtaGPX1, PtaGPX2, PtaGPX4, PtaGPX3, and C44A, C73A, C92A, and E80A mutants of PtaGPX3, respectively

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Using GSH as the reducing agent, the Pinus GPXs did not show any enzymatic activity toward H2O2, Cum-OOH, or t-BuOOH. In contrast, all proteins could reduce these peroxides using Trx as the reducing agent (Table 1). Among the four Pinus GPXs, PtaGPX1 and PtaGPX4 showed the highest and lowest catalytic activities toward H2O2, Cum-OOH, and t-BuOOH, respectively.

Table 1 Specific activities of the Pinus GPXs and mutants of PtaGPX3
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Because the four Pinus GPXs showed higher catalytic activities toward H2O2 than Cum-OOH or t-BuOOH, their kinetic constants were determined using H2O2 as the electron acceptor and Trx as the electron donor (Table 2). Compared with PtaGPX1 and PtaGPX3, PtaGPX2 and PtaGPX4 showed a much higher affinity (lower K m H2O2) for H2O2. PtaGPX2 and PtaGPX3 showed much higher affinity (lower K m Trx) toward substrate Trx than that of PtaGPX1 and PtaGPX4.

Table 2 Kinetic constants of the Pinus GPXs and E80A mutant of PtaGPX3
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Catalytic Properties of Glu80 Residue of PtaGPX3

According to previous molecular docking experiments, Glu80 of Populus PtGPX5 could form a hydrogen bond with Trx during the GPX reaction (Koh et al. 2007). Protein sequence alignment revealed that this residue was conserved in all plant GPXs (alignment position 149 in Fig. 1). The corresponding residue in PtaGPX3 was also Glu80. Therefore, the catalytic properties of Glu80 of PtaGPX3 were investigated using site-directed mutagenesis. When Glu80 of PtaGPX3 was replaced by alanine, the E80A mutant was expressed mainly as a soluble protein in E. coli at 37 °C. The purified mutant E80A protein showed specific activities toward the substrates H2O2, Cum-OOH, and t-BuOOH using Trx as the electron donor. Compared with the wild-type protein, the E80A mutant showed 6.1-, 24.5-, and 36.9-fold decreased catalytic activity toward H2O2, Cum-OOH, and t-BuOOH, respectively (Table 1). We also determined the apparent kinetic constants of the E80A mutant toward the substrates Trx and H2O2. Compared with the wild type, the E80A mutant showed a 3.1-fold higher affinity for Trx, but a 67.5-fold lower affinity for H2O2.

Catalytic Properties of the Three Conserved Cys in PtaGPX3

Multiple protein sequence alignments revealed that three Cys were conserved in all the plant GPXs (alignment positions 113, 142, and 161 in Fig. 1). Previous studies of Populus PtGPX3 demonstrated that two Cys residues (alignment positions 113 and 161 in Fig. 1) participated in the catalytic cycle and in Trx-mediated regeneration (Navrot et al. 2006). In this study, we selected PtaGPX3 to investigate the catalytic characterizations of the three conserved Cys residues in all plant GPXs using site-directed mutagenesis. Cys44, Cys73, and Cys92 of PtaGPX3 (corresponding to the three conserved Cys residues) were individually replaced with alanine. The C44A and C92A mutants were expressed mainly as soluble proteins in E. coli at 37 °C, whereas the C73A mutant was mainly insoluble. When the induction temperature was lowered to 20 °C, the C73A mutant became soluble. When Trx was used as the electron donor, the C44A and C92A mutants did not show any enzymatic activity toward substrates H2O2, Cum-OOH, and t-BuOOH, while the C73A mutant showed enzymatic activities toward these three substrates. Compared with wild type, the C73A mutant showed a higher catalytic activity toward H2O2 but reduced activity toward Cum-OOH and t-BuOOH (Table 1).

We examined the behavior of wild-type PtaGPX3 and its three mutants (C44A, C73A, and C92A) during denaturing polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and oxidizing conditions. Under reducing conditions (30 mM DTT), all the proteins migrated at their expected molecular mass of ∼19 kDa (Fig. 5, lanes 3, 5, 7, and 9). PtaGPX3 and its C44A and C73A mutants exhibited altered migration under oxidizing conditions (30 mM H2O2) (Fig. 5, lanes 2, 4, and 6), whereas the migration of the C92A mutant was unaffected (Fig. 5, lane 8).

Fig. 5
figure 5

Effects of redox conditions on wild-type and mutant PtaGPX3. The proteins were either oxidized with 30 mM H2O2 (lanes 2, 4, 6, and 8) or reduced with 30 mM DTT (lanes 3, 5, 7, and 9) and separated on 14 % SDS-PAGE

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Discussion

GPXs are key enzymes for scavenging ROS in plant cells. Previous studies of GPXs focused mainly on angiosperms. In this study, we cloned four GPXs from the gymnosperm P. tabulaeformis that shared high protein sequence identities with angiosperm GPXs. In animals, several GPXs have specific activities toward peroxide substrates using GSH as the reducing agent (Sies et al. 1997; Dimastrogiovanni et al. 2010; Barlow-Walden et al. 1995). In this study, we found that the four Pinus GPX proteins did not show enzymatic activity toward the substrates H2O2, Cum-OOH, and t-BuOOH using GSH as electron donor. But the four GPXs showed enzymatic activities toward peroxide substrates when Trx was used as the reducing agent. Other studies of angiosperm GPXs revealed that Trx was a better electron donor in the plant GPX reaction process than GSH (Iqbal et al. 2006; Jung et al. 2002). Therefore, plant GPXs might be thioredoxin-dependent peroxidases.

Although the four Pinus GPXs had high-sequence identities and similar expression patterns, they showed different enzymatic activities and kinetic characteristics. For example, PtaGPX1 had at least 5.0-fold higher activity toward the three substrates than PtaGPX4, whereas PtaGPX4 showed the highest affinity (lowest K m H2O2) for H2O2 among the four proteins. The differences in biochemical characteristics of the four Pinus GPX proteins indicated their functional divergence.

Previous molecular docking experiments proposed that a conserved Glu residue (alignment position 149 in Fig. 1) might form a hydrogen bond with Trx during the GPX reaction process. However, in this study, after removing the side chain of the Glu80 of PtaGPX3, the mutant showed much higher affinity toward Trx than the wild-type enzyme, suggesting that this conserved Glu residue might not be directly involved in the conjugation of GPX to Trx. We observed that, compared with wild-type PtaGPX3, the E80A mutant had much lower affinity toward H2O2, suggesting that Glu80 could affect the conjugation of PtaGPX3 to peroxides. In addition, compared to wild-type PtaGPX3, the E80A mutant showed much lower catalytic activities toward H2O2, Cum-OOH, and t-BuOOH, suggesting that this conserved Glu residue plays an important role in catalytic activity.

Previous studies proposed a three-step reaction mechanism for plant GPXs: (1) a nucleophilic attack of the catalytic Cys (Cys44 in PtaGPX3) on the peroxide and the concomitant formation of a sulfenic acid, (2) an attack of the sulfenic acid by a Cys (Cys92 in PtaGPX3) and formation of an intramolecular disulfide bridge between these two Cys, and (3) the reduction of the disulfide bridge by Trx (Navrot et al. 2006; Koh et al. 2007). These two Cys were absolutely conserved in all the plant GPXs assessed (alignment positions 113 and 161 in Fig. 1). The crystal structure of Populus PtGPX5 also showed that these two conserved Cys residues could form an intramolecular disulfide bond under oxidizing conditions (Koh et al. 2007). In this study, we used the crystal structure of PtGPX5 under oxidizing conditions to model the 3D structure of PtaGPX3. The modeled structure of PtaGPX3 revealed that Cys44 and Cys92 could form an intramolecular disulfide bond (Fig. 3c). PtaGPX3 contained only three Cys residues: Cys44, 73, and 92 (alignment positions 113, 142, and 161 in Fig. 1). When removing the side chain of the Cys73 of PtaGPX3, we observed a redox-dependent shift of the mutant in SDS-PAGE, suggesting that an intramolecular disulfide bond had formed between Cys44 and Cys92 under oxidizing conditions. In addition, after removing the side chain of the Cys44 or Cys92 of PtaGPX3, the mutants completely lost catalytic activity to the peroxides, suggesting that these two Cys residues were critical for catalytic activity.