Two Wheat Glutathione Peroxidase Genes Whose Products Are Located in Chloroplasts Improve Salt and H2O2 Tolerances in Arabidopsis

Oxidative stress caused by accumulation of reactive oxygen species (ROS) is capable of damaging effects on numerous cellular components. Glutathione peroxidases (GPXs, EC 1.11.1.9) are key enzymes of the antioxidant network in plants. In this study, W69 and W106, two putative GPX genes, were obtained by de novo transcriptome sequencing of salt-treated wheat (Triticum aestivum) seedlings. The purified His-tag fusion proteins of W69 and W106 reduced H2O2 and t-butyl hydroperoxide (t-BHP) using glutathione (GSH) or thioredoxin (Trx) as an electron donor in vitro, showing their peroxidase activity toward H2O2 and toxic organic hydroperoxide. GFP fluorescence assays revealed that W69 and W106 are localized in chloroplasts. Quantitative real-time PCR (Q-RT-PCR) analysis showed that two GPXs were differentially responsive to salt, drought, H2O2, or ABA. Isolation of the W69 and W106 promoters revealed some cis-acting elements responding to abiotic stresses. Overexpression of W69 and W106 conferred strong tolerance to salt, H2O2, and ABA treatment in Arabidopsis. Moreover, the expression levels of key regulator genes (SOS1, RbohD and ABI1/ABI2) involved in salt, H2O2 and ABA signaling were altered in the transgenic plants. These findings suggest that W69 and W106 not only act as scavengers of H2O2 in controlling abiotic stress responses, but also play important roles in salt and ABA signaling.


Introduction
In higher plant cells, several metabolic processes (e.g. photosynthesis and respiration) and adverse environmental conditions (e.g. high-salt, drought, mechanical stimulation, chemical toxicity, pathogen infections and extreme temperatures) promote generation of reactive oxygen species (ROS), including hydroxyl radicals (OH), superoxide radicals (O 2 ?2 ), and hydrogen peroxide (H 2 O 2 ) [1][2][3][4][5]. ROS are capable of causing damage to membrane lipids, proteins and nucleic acids and these highly reactive molecules are believed to be the major contributing factors in causing rapid cell damage [6]. Exogenous H 2 O 2 induces the expression of defense genes as well as initiating programmed cell death in plants [7,8]. The steady-state level of ROS is mainly determined by the activity of antioxidant system in plant cells [9]. Several endogenous antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) play important roles in protecting plants cell from oxidative injury [10]. GPX is a key enzyme for scavenging H 2 O 2 in plant cells [11].
As an antioxidant enzyme, GPX in mammals reduces H 2 O 2 and organic hydroperoxides to water and correspondingly alcohols using glutathione (GSH) to protect cells from oxidative damage [12,13]. Based on their amino acid sequences, substrate specificity, and tissue localization, the GPX family in mammals can be divided into five classes, cytosolic GPX (GPX1), gastro-intestinal GPX (GPX2), plasma GPX (GPX3), phospholipid hydroperoxide GPX (PHGPX, also called GPX4), and seleno-independent epididymis GPX (GPX5) [12,14]. Among GPX isoforms, vertebrate GPX5 proteins lack the selenocysteine residue replaced by a cysteine residue in the catalytic site [15,16]. The selenocysteine residue in its presumed catalytic site is important for the catalytic activity of GPXs, but the replacement of selenocysteine by cysteine greatly reduces the activity of enzymes in animals [17]. PHGPX is an antioxidant selenoenzyme present in a variety of adult and embryonic tissues. It is the principal basis of a defense system that intimately participates in the repair of disrupted biomembranes by interacting directly with peroxidized phospholipids in biomembranes [18][19][20][21].
The plant GPX family shares highest sequence homology to animal PHGPXs, rather than to any of the other members of the animal GPX family [22][23][24][25][26]. These proteins from plants possess a Cys residue, rather than selenoCys, in their presumed catalytic site, suggesting that GPX in plant is not a Se-dependent protein [27]. However, Amino acid replacement of the catalytic selenoCys by Cys results in a relatively low activity in plants compared to homologous animal GPXs [28]. GPXs are ubiquitously occurring enzymes in subcellular organelles, including the cytosol, nucleus, chloroplast, mitochondria, and peroxisome, where GPXs use GSH or thioredoxin (Trx) as a reducing agent to reduce H 2 O 2 , organic hydroperoxide, and lipid hydroperoxides [2,12,[29][30][31][32]. Plant GPX isoenzymes display an obvious preference to Trx [24,33].
Besides GPXs another group of enzymes capable of scavenging peroxides in plants have been studied extensively. It is known that certain plant glutathione-S-transferase (GST) displayed also strong GPX activity (GST/GPX) toward organic hydroperoxides catalysing their reduction to the less toxic alcohols [34].
Plant GPXs are involved in responses to many abiotic stresses and hormones [27,[35][36][37]. A rice GPX gene was strongly expressed in normal tissues of high photosynthetic efficiency as well as in tissues stimulated by oxidative stresses [38]. Overexpression of the tobacco GST/GPX gene confers salt and chilling tolerance due to enhanced ROS scavenging and reduces membrane damage in transgenic tobacco plants [10]. It was demonstrated that Arabidopsis GPX3 plays dual roles in H 2 O 2 scavenging and ABA signal transduction [39]. Furthermore, chloroplastic GPXs in Arabidopsis play a role in cross talk between photooxidative stress and immune responses [5,40]. As has been reported, expressing a tomato PHGPx in tobacco could prevent Bax induced cell death, indicating the role of GPX in PCD in plant [41,42].
Wheat (Triticum aestivum L.) is one of the most important cereal crops in the world; however, ROS accumulation in cells induced Oryza sativa (OsGPX01, accession number: NC008397), Zea mays (ZmGPX01, AY542310) and Homo sapiens (HsGPX04, P36969). Gray background represents strictly conserved amino acids. Boxed sequences represent highly conserved domains (G1,G2,and G3). The SeCys residues of the mammalian PHGPXs (HsGPX04) are denoted by ''U'', the three conserved Cys of these isoenzymes are marked by inverted triangles. (B) Exon-intron structure of the GPX homologs. The chromosomal structures of the GPXs were constructed by comparing mRNA sequences with their respective genomic sequences. The length of each exon (square) and intron (line) is given. The W69 gene starts with ATG at position 75 bp in exon 1 and ends with TAG at position 81 bp in exon 6; the W106 gene starts with ATG at position 56 bp in exon 1 and ends by TGA at position 29 bp in exon 6. doi:10.1371/journal.pone.0073989.g001 by environmental stresses can cause severe oxidative damage to wheat growth and grain yield [43]. At present, there is no report on the role of GPXs in protecting wheat plants from oxidative damage. In this study, we isolated two wheat cDNAs encoding GPX proteins for the first time by de novo transcriptome assembly technology. The isoenzymatic characteristics, subcellular localization, and transcript accumulation of the recombinant protein when transgenic plants were subjected to different stresses were investigated.

Isolation and characterization of W69 and W106
We generated an extensive expressed gene catalog for salttreated wheat using Illumina mRNA Sequencing technology and de novo assembly (unpublished data). Two up-regulated putative GPX cDNA fragments, designated as W69 (GenBank Accession No. KF031945) and W106 (GenBank Accession No. KF031946), were selected from the total transcript-derived assembled unigenes. Full-length cDNA of W69 and W106 were cloned successfully from wheat cDNA by reverse transcription PCR (RT-PCR) using special primers sets. The predicted W69 protein consists of 214 amino acids with a calculated molecular mass of 21.3 kD, and W106 protein consists of 211 amino acids and calculated molecular mass of 19.2 kD. Multiple sequence alignment of W69 and W106 with other reported GPXs from animals and plants showed that some motifs of GPXs were conserved among different species (Fig. 1A). Three completely conserved motifs GKVLLIVNVASRCG (GPX signature 1), LAFPCNQ (GPX signature 2), and WNF(S/T)KF) existed within most plant and mammalian GPX sequences. Cysteine residues coded by UGU located in the ''GPX signature 1'' are the specific structure of selenium-independent plant GPXs, whereas selenium-dependent HsGPXs from mammals possess a seleno-cysteine residue coded by UGA (Fig. 1A). A phylogenetic tree of GPXs from plants revealed that GST family can be classified into five classes. W69 and W106 belong to one group located to chloroplasts (Fig. 2B).
Isolation of the genomic sequences of the wheat GPXs revealed both W69 and W106 comprised six exons and five introns (Fig. 1B). All of the exon/intron splice junctions in W69 DNA sequence conform to the canonical GT/AG boundary [44], intron 5 of W106 gene has a GT/GC sequence.
To gain insight into the mechanism responsible for transcriptional regulation, we isolated a 1,862 bp promoter region upstream of the W69 ATG start codon, and 1,600 bp of W106 from genomic DNA using a PCR strategy. We searched for putative cis-acting elements in the promoter regions using the database Plant cis-acting Elements, PLACE (http://www.dna. affrc.go.jp/PLACE/) ( Table 1). A number of regulatory elements responsive to drought, salt, low-temperature, and ABA were recognized, including ABRE, DRE, DRE/CRT, and DPBF binding sequences. In addition, a gibberellin responsive element (GARE) and an ethylene responsive element (ERE) were identified. More importantly, the W69 and W106 promoter regions have antioxidant-responsive elements, ARE, which was found in plant and human promoters (Table 1).

Expression patterns of W69 and W106 under stress
The W69 and W106 promoter regions have multiple abiotic stress-responsive elements (Table 1). To assess responses to abiotic stresses, we quantitatively surveyed the transcript expressions of W69 and W106 using quantitative real-time PCR (Q-RT-PCR). As shown in Fig. 3, both transcripts began to respond to environmental stress at early stages, but exhibited different expression patterns.
Both W69 and W106 were rapidly activated to peak within 0.5 h after salt treatment, then declined to normal levels after 1 h, and were barely detectable within 24 h. Both were also rapidly induced by drought and ABA, reached a maximum level at 4 h and 1 h, respectively, and then declined. W69 was strongly induced by drought, but only weakly by ABA. In contrast, W106 was highly activated by ABA, but weakly by drought. In addition, W106 was immediately activated by H 2 O 2 and absent after 6 h; however, the transcript of W69 was barely detectable over a 24 h treatment with H 2 O 2 . These results implied that the GPX subfamily may function mainly in oxidative responses induced by salt, H 2 O 2 and ABA stresses, but with some divergence in roles between W69 and W106.

GPX activities of W69 and W106 in vitro
Overexpression of recombinant GPXs in E. coli BL21 cells was observed after IPTG induction. Samples of total proteins and purified soluble protein fractions were separated on SDS-PAGE. W69 (21 kDa) and W106 (19 kDa) were successfully expressed (Fig. 4A).
The purified protein was prepared for enzymatic analysis. Results showed that the recombinant isoenzymes could catalyze H 2 O 2 and t-BHP using E. coli Trx or GSH as electron donors. However, the isoenzymes reduced hydroperoxides with Trx at a higher rate than with GSH, indicating that Trx was likely the favored electron donor (Fig. 4B). In contrast to t-BHP, the isoenzymes exhibited a higher affinity for H 2 O 2 and GPX activities of W69 were detected at a higher level than W106 utilizing Trx, which is the main electron donor during redox reactions. As expected, the pET-28A-transformed control had no obvious GPX activity (Fig. 4B).

Chloroplast localization of W69 and W106
GPX proteins in plants were found in the cytosol, chloroplast, and mitochondria. The PSORT system (http://www.psort.org/) program for predicting subcellular localization, indicated that both W69 and W106 with a chloroplastic N-terminal transit peptide would most probably localize in chloroplasts. To investigate the biological activity of the W69 and W106 proteins, the full-length cDNAs of W69 and W106 were fused in frame with the green fluorescent protein (hGFP) gene under the control of the CaMV 35S promoter, and transferred into wheat mesophyll protoplasts. Fluorescence of both W69-GFP and W106-GFP was specifically detected in chloroplasts, whereas the control expressing hGFP alone showed fluorescence distributed throughout the protoplasts (Fig. 5). Given their localization in chloroplasts, possible roles for W69 and W106 would be in protection of chloroplasts against oxidative damage induced by photosynthesis or external stresses [3].

Improved salt tolerance in transgenic Arabidopsis
GPX genes are important in detoxification of many cellular degradation products formed during oxidative stress. Additionally, GPXs could impart stress tolerance through their peroxidase activities [45]. To further investigate the biological functions of the two GPXs, two T3 generation transgenic Arabidopsis lines were chosen to examine effects on germination and growth under salt stress. Transgenic and wild-type Arabidopsis seeds were grown on MS medium for 3 d at 22uC, and then transferred on MS medium containing different NaCl concentrations (50-200 mM).
Transgenic W69 and W106 seedlings had similar phenotypes to wild-type seedlings under normal conditions. As expected, salt stress reduced the growth of both transgenic seedlings and wildtype seedlings to some extent (Fig. 6). At 150 mM NaCl, transgenic plants remained green, whereas wild-type plants displayed chlorosis and growth inhibition after 15 days. The root lengths of W69-7 lines were almost 4-fold those of wild-type plants (Fig. 6A). Furthermore, transgenic W69 seedlings were more tolerant of salt stress than seedlings of W106 at different NaCl concentrations (Fig. 6B). In addition, the transgenic Arabidopsis plants showed higher germination rates than wild-type under high salt stress. Transgenic seeds germinated in up to 200 mM NaCl, whereas wild-type seeds failed to germinate under the same conditions (data not shown). Therefore, overexpression of W69 and W106 resulted in enhanced early tolerance to high salt stress. . Phylogenetic tree of W69 and W106 with other GPXs from plants. All GPXs proteins were clustered using ClustalX, and the phylogenetic tree was generated by MEGA5 using the neighbor-joining algorithm. In addition to the sequences of W69 and W106, the tree was constructed using sequences from Arabidopsis thaliana: AtGPX01, At2g25080; AtGPX02, At2g31570; AtGPX03, At2g43350; AtGPX04, At2g48150; AtGPX05, At3g63080; AtGPX06, At4g11600; AtGPX07, At4g31870; AtGPX08, At1g63460.  We also examined tolerance to drought stress in the transgenic plants, but observed no detectable increases in tolerance (data not shown).

Transgenic Arabidopsis has increased tolerance to H 2 O 2
Three-day-old seedlings of transgenic and wild-type Arabidopsis were transferred to MS medium plates supplemented with 1 mM H 2 O 2 . At 15 d, the seedlings of W69 and W106 grew vigorously and were larger than wild-type seedlings. At 1 mM H 2 O 2 , the transgenics had longer primary roots than wild-type plants. There were also significant increases in the number and total lengths of the lateral roots compared to wild-type (Fig. 7A). As shown in Fig. 7B, H 2 O 2 treatment significantly decreased the fresh and dry weights of transgenic and wild-type plants, but the transgenics were less affected by H 2 O 2 treatment than wild-type plants. In addition, leaves of wild-type plants tended to become yellow during growth under H 2 O 2 treatment whereas those of transgenic plants remained green (Fig. 7A). Thus the transgenic seedlings apparently had increased peroxide-scavenging capacity and enhanced tolerance to H 2 O 2 .
Germination of W69 and W106-overexpressed seeds is insensitive to ABA To investigate the effect of ABA on germination of W69and W106-overexpressing seeds, we planted seeds on MS medium containing different ABA concentrations. As shown in Fig. 8A, there was no difference in seed germination between the wild-type and transgenic plants under normal conditions. At 0.8 mM ABA there was some inhibition of germination of both wild-type and transgenic seeds, but the latter were affected to a lesser extent. Approximately 42% of W69-7 overexpressed seeds and 39% of W106-2 overexpressed seeds developed cotyledons and became green compared with only 10% wild-type germination after 6 days (Fig. 8B). Addition of 1 mM ABA entirely arrested the emergence of cotyledons of both transgenic and wild-type seeds (Fig. 8B). These results indicated that GPX overexpression relieves the ABAmediated inhibition during seed germination and early seedling growth.
W69 and W106 displayed changed stress-responsive gene expressions SOS1, RbohD and ABI1/ABI2 are key regulators in response to salt, oxidative stress and ABA, respectively [39,[46][47][48]. To determine whether stress response pathways were affected by the GPX overexpression, we used Q-RT-PCR to analyse these genes in wild-type and transgenic Arabidopsis. Overexpression of W69 and W106 indeed changed the transcript levels of these marker genes. For example, W69 activated SOS1 and RbohD expression to levels about 1.8-and 1.5-fold higher, respectively, than those in wild-type. In contrast, the expression levels of SOS1 and RbohD were only slightly higher in W106 seedlings than in wild-type. Furthermore, ABI1 and ABI2 transcript levels in both transgenic lines appeared to be slightly lower than in wild-type (Fig. 9). These findings suggest that these GPXs may be signaling components in the ROS, ABA and salt-response pathways.

Discussion
Animal GPXs are 20-22 kD monomer proteins, that can directly reduce phospholipid hydroperoxides, fatty acid hydroperoxides, and cholesterol hydroperoxides produced in peroxidized membranes [18]. Animal GPXs are considered crucial for protecting membranes from oxidative stress. Though GPX-like cDNAs had been found in a diversity of plants [22,23,38,[49][50][51], their physiological functions in plants had not been clearly resolved. Plant GPXs belong to a small GPX multigenic subfamily involved in response to various environmental stresses and had been considered to be the main defense strategy against oxidative membrane damage by detoxifying organic hydroperoxides as well as H 2 O 2 [31,[52][53][54][55].
In the present study, we describe the identification and function of two wheat GPXs, designated W69 and W106, that share relatively high levels of sequence similarity with other plant GPXs and human GPX04 (Fig. 1). Genomic sequences analysis of the two GPXs revealed that both comprise six exons and five introns. The nucleotide sequences and sizes of exons 2-5 of the isoenzymes genes were very similar, although the nucleotide sequences and sizes of the corresponding introns were highly divergent. These results provided evidence supporting tightly conserved exon-intron structures of GPX homologs across the plant kingdom [13]. In particular, there was a high degree of conservation among plant species in the lengths of exons 2-5, whereas the lengths of exons 1 and 6 were quite divergent [56]. The homologous human HsGPX04 gene contains seven exons. Plant and mammalian GPXs have very divergent genomic organization patterns and analogous functions, reinforcing the hypothesis of an independent evolutionary scenario accompanying the structural diversification but functional similarity [13].
Salinity stress, mainly caused by NaCl and a serious threat to crop productivity, causes osmotic and ionic imbalances in cells [57]. On the other hand, salinity increases production and accumulation of ROS, which affects cell redox homeostasis and causes oxidative damage [58]. In the present work, we showed that overexpression of W69 and W106 increased the seed germination rate and boosted seedling growth in transgenic Arabidopsis plants during exposure to salt stress conditions (Fig. 6). In addition, exposure of wheat seedlings to NaCl or H 2 O 2 induced rapid accumulation of W69 and W106 transcripts (Fig. 3). Therefore, it was likely that increased mRNA accumulation in W69 and W106 was an early response in ROS reduction or a manifestation of adjustment to environmental stress [59]. H 2 O 2 is a central signaling molecule in stress and wounding responses, pathogen defense, and regulation of the cell cycle and cell death. Exogenous applications of H 2 O 2 promote the formation and development of adventitious roots in seedlings, but a relatively higher concentrations H 2 O 2 and longer times of exposure lead to inhibitory effects on rooting [60,61]. Measurement of GPX activities showed that the two wheat GPXs can catalyze H 2 O 2 or t-BHP in vitro. High level of GPX involved in the scavenging of H 2 O 2 in plants helps to protect against H 2 O 2 -induced tissue injury and increases tolerance to oxidative damage induced by salt or ROS [62,63]. Under exogenous H 2 O 2 treatment, we found that transgenic plants became stronger than wild-type. In addition, the transgenic seedlings had greater total lateral root lengths than wild-type and improved tolerance to ROS stress (Fig. 7). W69 and W106 were located in the chloroplasts (Fig. 5), cell components with the highest production sites of ROS [64,65]. These results indicated that overexpression of W69 and W106 conferred enhanced antioxidant capacity and protected plants against oxidative damage.
ABA is a phytohormone controlling seed dormancy and preventing seed germination during early embryogenesis. It also plays an essential role in stress response to drought, cold, and oxidative and osmotic stresses in the vegetative growth phase [66,67]. Some reports indicate that ABA can trigger the production of H 2 O 2 [68][69][70]. Arabidopsis seeds overexpressing W69 and W106 displayed higher germination rates than wild-type after ABA treatment, suggesting that transgenic seedlings have weakenedsABA control due to the scavenging effects of H 2 O 2 (Fig. 8). Therefore, W69 and W106 might play an important role in the ABA signal transduction. It was reported that mutation of AtGPX3 affected the expression of ABA-responsive genes and that the AtGPX3 gene might act as an oxidative signal transducer in ABA and drought stress signaling [71]. Q-RT-PCR indicated that overexpression of W69 and W106 also affected the transcriptional levels of key regulator genes involved in ABA, H 2 O 2 and salt signaling. This was in agreement with the important role of the GPX gene family in responding to environment stresses. Analysis of upstream cis-regulating elements of W69 and W106 provided a way for us to understand how GPX expression was controlled. For instance, ABRE and other ABA-related elements (such as the DRE and DPBF binding sites) found in the promoters of W69 and W106 might be responsible for the responses of genes to ABA. The CRT/DRE and GT1GMSCAM4 elements present in the promoters of the two isoenzymes might be responsible for genetic responses to NaCl. More importantly, ARE, an antioxidant-responsive element in rat is also present in the promoters of the two isoenzymes, and might be related to genetic response to oxidative stress (Table 1). Collectively, W69 and W106 were not only participated directly in ROS signaling, but were also involved in ABA and salt signaling cascades.
In summary, overexpression of W69 and W106 in Arabidopsis seedlings provided protection from salinity stress during germination and seedling growth. This protective effect appeared to allow transgenic plants to retain high levels of metabolic activity and growth due to increased GPX expression and reduced levels of oxidative damage compared to wild-type seedlings. Our study should be beneficial for obtaining detailed insights into the complex function of plant GPXs, and for analyzing specialized physiological characteristics of this antioxidant system in other organisms [19]. The GPXs might be excellent candidates for genetical engineering of crop plants with improved salt tolerance.

Plant materials and stress treatments
Wheat (T. aestivum cv. Xiaobaimai) seedlings grown hydroponically at 25uC for 10 d were subjected to various abiotic stresses. Seedlings were exposed to air on filter paper for rapid induction of water stress. To mimic salinity and ABA treatments, roots were submerged in 100 mM NaCl or 100 mM ABA, respectively. For hydrogen peroxide treatment, seedlings were transferred to solutions containing 0.1 mM H 2 O 2 . Materials were collected at 0, 1, 2, 4, 16, 12, and 24 h after various treatments. Harvested plants were dropped immediately into liquid nitrogen and stored at -80uC for RNA extraction.

General bioinformation
Putative amino acid sequences used in this study were from GenBank databases. Conserved motifs were investigated by multiple alignments using DNAMAN version 6.0. Phylogenetic trees were constructed with ClustalX using the neighbor-joining (NJ) method.

Quantitative real-time PCR
Expression patterns for the two GPX genes were measured by Quantitative real-time PCR (Q-RT-PCR), using the ABI Prism 7300 system (Applied Biosystems, USA). Total RNA was extracted from young leaves of 2-week-old wheat plants using Trizol reagent according to the manufacturer's protocol (TianGen, Beijing, China) and DNase I digestions were applied (TaKaRa, Japan). First-strand cDNA was synthesized with AMV Reverse Transcriptase (TaKaRa, Japan). Each PCR was repeated three times in total volumes of 20 ml containing primer (4 mM), cDNA (40 ng) and 9 ml RealMasterMix (TianGen, Beijing, China). Quantification of the target gene expression under each stress was carried out by the relative 22ggCT method [72]. The actin gene was used as an internal control for normalization of template cDNA. The gene-specific primers were as follows: W69, 5'-ACAGAG-GATTCGGGAGGACT-3' and 5'-AGGAGGACCTTCCC-CTTGTA-3'; W106, 5'-ACAAGGGGAAAGTCCTGCTT-3' and W106-RTR 5'-CTGGTTCCTGTCCAGCAAAT-3'; wheat actin, 5'-CTCCCTCACAACAACCGC-3' and 5'-TACCAG-GAACTTCCATACCAAC-3'. Q-RT-PCR was also performed to analysis the expression of stress-and ABA-responsive genes, using 2-week-old transgenic and wild-type Arabidopsis seedlings. The actin gene was used as an internal control. The specific primers were as follows: RbohD  After growing in MS media with added H 2 O 2 (1 mM) for 15 d after germination, seedlings were collected and analysed. Data are given for the length of the primary root, lateral root number, total lateral root length, seedling fresh weight and dry weight. Data are means 6SD of three independent experiments. Asterisks indicate a significant difference (*P,0.05; Student's t-test) relative to wild-type control. doi:10.1371/journal.pone.0073989.g007 Actin2 in Arabidopsis, 5'-GGTAACATTGTGCTCAGTGGTG-3' and 5'-C GACCTTAATCTTCATGCTGC-3'.

Detection of enzyme activities of the GPX isoenzymes
The two GPX cDNAs were amplified by reverse transcription PCR (RT-PCR) using the primer sets W69-PET (5'-TACG-GATCCATGGGGGCGTCCGAATCT-3' and 5'-TAGCTC-GAGCTTCTGCGAGTCGGAAGATTCC-3') and W106-PET (5'-TACGGATCCAACATGGGTGCGGCAGAGT-3' and 5'-TAGCTCGAGAACCTCCAACAGCTTCTTGATG-3'). The PCR product was cut by BamHI and XhoI and then subcloned into corresponding sites of pET-28a (Novagen) with a Histidinetag at the N-terminus. The fusion plasmids and empty vector (PET-28a) were transformed into E. coli BL21 (DE3) strain. E. coli cells carrying pET-W69, pET-W106, and PET-28a were grown in 40 ml LB medium containing 50 mg/ml kanamycin at 37uC to D 600 = 0.5 and induced with 0.8 mM isopropyl-1-thio-bDgalactopyranoside (IPTG) at 22uC. Cells were extracted at 4 h and were collected by centrifugation at 3000 g for 10 min, resuspended in pre-chilled sodium phosphate buffer (14 mM NaCl, 0.27 mM KCl, mM Na 2 HPO 4 , 0.18 mM KH 2 PO 4 , 1 mM EDTA-2Na and 1% polyvinylpyrrolidone, pH 7.0), and disrupted by sonication. His-tag fusion pET-W69/pET-W106/and/PET-28a proteins were purified using Ni-NTA Agarose (Qiagen, Tokyo, Japan) according to the manufacturer's instructions.  . Expression effects of ABA-and stress-responsive genes in W69 and W106 Arabidopsis using Q-RT-PCR. Total RNA was isolated from leaves of 2-week-old Arabidopsis seedlings. The actin2 gene was used as an internal reference. Relative expression of W69 and W106 were normalized to the transcript abundances in wild-type controls (normalized as 1). Error bars represent standard deviation among three biological replicates. Asterisks indicate a significant difference (P,0.05; Student's t-test) relative to wild-type control. doi:10.1371/journal.pone.0073989.g009 Protein concentrations were quantified using the Bradford method. Purified proteins from each sample were loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis.
Enzyme activity was measured spectrophotometrically using a Glutathione Peroxidase Assay Kit (BioAssay systems, USA) with minor modifications. E. coli Trx and t-BHP were purchased from Sigma Aldrich (St. Louis, MO, USA). The reaction was started by addition of H 2 O 2 or t-butyl hydroperoxide. The absorbance was immediately read at OD 340 nm and again at 4 min using a Perkin-Elmer Lambda 35 UV/VIS spectrometer (Perkin-Elmer Instruments, USA). The unit activity (U/L) is the amount of GPX that produces 1 mM of oxidized glutathione (GSSG) per min at pH 7.6 and room temperature.

Transient expression of green fluorescent protein-fused GPXs
Green fluorescent protein (GFP)-fused GPXs, coexpression plasmids was constructed for the expression studies. The full open reading frames of the W69 and W106 genes were respectively cloned into 16318hGFP vectors, fused with the GFP reporter gene under the control of the 35S promoter [73]. The recombinant W69-hGFP and W106-hGFP fusion plasmids were transformed to common wheat mesophyll protoplasts by the PEG-mediated method [74]. The expression of fusion proteins was monitored after 12 h of incubation in a dark room, and images were captured under a laser scanning confocal microscope (Zeiss LSM700, Germany). GFP 'uorescence was collected in the 500-570 nm wavelength range. For chloroplast auto'uorescence, the wavelength range was 630-700 nm.

Generation of transgenic Arabidopsis lines with GPX genes
Fragments of W69 and W106 were separately ligated into the modified pBI121 vector under control of the CaMV 35S promoter [75]. Kanamycin-resistant Arabidopsis transformants carrying W69 or W106 were generated using the vacuum infiltration method. Transformed plants were cultured on Murashige and Skoog (MS) medium containing 0.8% agar and 50 mM Kanamycin in a day/ night regime of 16/8 h under white light (50 photons m 21 s 21 ) at 22uC for 2 weeks and were then transferred to soil. The T3 generation seeds obtained were used for further analysis.

Stress treatments
Seeds of transgenic (T3 generation) and wild-type Arabidopsis plants were grown on 10610 cm MS agar plates. They were routinely kept for 3 days in the dark at 4uC to break dormancy and transferred to a tissue culture room under a day/night of 16/ 8 h under white light (with 50 photons m 21 s 21 ) at 22uC for 3 d.
For salt treatment, 7-day-old seedlings were transferred to MS agar plates containing 150 mM NaCl for 7 days. For drought treatment, the seedlings were placed on MS agar plates with mannitol (100-200 mM) for 7 days. For H 2 O 2 treatment, the plants were placed on MS agar plates supplemented with H 2 O 2 (1-5 mM) for 7 days. For ABA treatment, the seeds of transgenic and wild-type Arabidopsis were grown on MS agar plates supplemented with 0.5-1 mM ABA for 7-14 days. Seeds were considered germinated when radicles had completely emerged from the seed coat.
The root lengths of Arabidopsis were measured with a ruler, and lateral roots were counted and measured with a dissection microscope. The fresh weight was measured on an analytical balance immediately after plant harvest. The samples used for dry weight determinations were measured after 48 h at 70uC.