Proteomic profiling analysis reveals that glutathione system plays important roles responding to osmotic stress in wheat (Triticum aestivum L.) roots

Plant materials and the measurement of relative water content (RWC)

Seeds of Aikang58 were sterilized using 0.1% HgCl₂ for 7 min and washed eight times with sterile distilled water. Subsequently, the seeds were cultured in Petri dishes in a chamber under the same conditions according to Li et al. (2013). At the two-leaf stage, the wheat seedlings were transferred into Hoagland solution containing 0%, 5%, 10%, 15% and 20% PEG-6000 to simulate osmotic stress. After cultivation for 24 h, the root tissues from seedlings exposed to the five treatments were collected and frozen in −80 °C for subsequent experiments. RWC was measured according to Gao et al. (2011).

Protein extraction

Frozen root samples were thoroughly ground into powder in liquid nitrogen. Lysis buffer (pH 8.5), containing 2 M thiourea, 7 M urea and 4% CHAPS with protease inhibitor (Sigma, USA), was added to the powder at 1:10 (w/v). The mixture was sonicated for 60 s and extracted for 30 min at room temperature. Subsequently, the mixture was centrifuged at 40,000 g for 1 h at 10 °C, and the supernatant was transferred to a 50 mL tube containing four volumes of 10% (w/v) TAC/acetone. After mixing, the mixture was stored at −20 °C overnight, and the supernatant was removed after centrifugation at 40,000 g for 10 min at 4 °C. The protein was washed three times with acetone and then dried through lyophilization to form a protein powder, and suspended in lysis buffer (2 M thiourea, 7 M urea and 4% CHAPS). The protein concentration was determined using the Bradford assay with BSA as a standard. The remaining samples were stored at −80 °C until further use.

Trypsin digestion and iTRAQ labeling

All reagents and buffers for iTRAQ labeling and cleaning were purchased from Applied Biosystems (Foster City, CA, USA). iTRAQ labeling was performed according to the manufacturer’s instructions. The proteins were dissolved, denatured, alkylated and digested with trypsin at 37 °C overnight. And 100 µg of the digestion product were thawed and reconstituted in 150 µL of isopropanol, and subsequently labelled with iTRAQ reagent (Applied Biosystems). The iTRAQ experiment just contained two experiment settings of four-plex and eight-plex, which could only analyze four or eight samples once time respectively. In previous studies, researchers had performed the eight-plex iTRAQ experiments using the setting of 3:3:2 and 3:2 (Longworth et al., 2012; Ge et al., 2014). In the present study, the experiment setting of 3:3:2 (eight-plex) was selected for this analysis. The three biological replicates of roots exposed to 15% PEG-6000 treatment were labeled with 113, 115 and 121 tags, the three biological replicates of roots exposed to 10% PEG-6000 treatment were labeled with 114, 118 and 119 tags, and the two biological replicates of control (0% PEG-6000) were labeled with 116 and 117 tags (Fig. 1). Subsequently, the labeled samples were pooled in equal ratios. The labeled peptide mixture was dissolved in 100 µL mobile of phase A (2% (v/v) acetonitrile, 98% (v/v) ddH2O, pH 10) and subsequently centrifuged at 14,000 g for 20 min. The supernatant was carefully collected and further loaded onto the column for stepwise elution through the injection of mobile phase B (98% acetonitrile, 2% ddH2O, pH 10) with a 700 µl/min flow rate. The fractions were eluted (1.8 min each) and collected using step gradients of mobile phase B.

Analysis using Q-Exactive mass spectrometer

The fractionated peptides were analyzed using a Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) fitted with a nano-liquid chromatography system (Thermo Scientific EASY-nLC 1000 System). A binary solvent system comprising 99.9% H2O, 0.1% formic acid (phase A) and 99.9% ACN, 0.1% formic acid (phase B) were used to elute the peptides. The following linear gradient was used: 4–8% B in 5 min, 8–35% B in 35 min, 35–90% B in 5 min, washed at 95% B for 6 min, and equilibrated with 4% B for 8 min at a 350 nL/min flow rate. The eluent was further introduced to a Q-Exactive mass spectrometer via an EASY-Spray ion source. The following source ionization parameters were used: 2.1 kV spray voltage, capillary temperature 250 °C and 100 V declustering potential.

A Top 20 data-dependent mode with automatic switching between MS and MS/MS was used in mass spectrometer. Full-scan MS mode (350–1,800 m/z) was performed at a resolution of 70,000 with 1 × 10⁶ ions automatic gain control (AGC) target and a maximum ion transfer (IT) of 60 ms. The precursor ions were fragmented using high-energy collisional dissociation (HCD) and subjected to MS/MS scans with the following parameters: 17,500 resolution, AGC with 5 × 10⁶ ions, maximum IT with 70 ms, 5,000 intensity threshold and 29% normalized collision energy.

Sequence database searching and data analysis

Mascot 2.2 (Matrix Science, London, UK) and Proteome Discoverer 1.4 (Thermo Electron, San Jose, CA) were used for processing the raw data of MS/MS spectra and completing database search and a quantitative analysis against a non-redundant protein database of hexaploid wheat genome, which had been generated by Mayer et al. (2014) and provided as File S1. For database searching, the following parameters were used: trypsin enzyme, two missed cleavages at maximum, 20 ppm of peptide mass tolerance, 0.1 Da of fragment mass tolerance, carbamidomethylation of cysteine as fixed modification, methionine oxidation and iTRAQ 8 plex labels at the N-termini and at lysine side chains as dynamic modification. For protein identification, only peptides with significant scores (iron score ≥ 35) at 99% confidence interval were used, and 2,228 proteins were finally got, of which 1,391 proteins with two or more peptides were considered for further analysis. The protein fold-change was obtained based on the quantity comparison between each treatment sample and the average level of control. For statistical analysis, the average fold-change ≥ 95% confidence interval and P-values ≤ 0.05, which was got by the t-test with different repeat times in two groups, were considered significant. The sequence data of the DEPs was searched against the UniProt database for protein function, and the BlastKOALA website (http://www.kegg.jp/blastkoala/) was used for the KEGG analysis with an E-value of 1 × 10⁻⁵.

The mass spectrometry data have been deposited to the iProx database with the accession number: IPX00075800.

Phylogenetic analysis of glutathione S-transferases (GSTs)

Multiple amino acid sequence alignment of GSTs was performed using ClastalW. An unrooted phylogenetic tree of these GST protein sequences was constructed using the neighbor-joining method with MEGA 5.10 software, and a bootstrap analysis with 1,000 replicates was performed to assess the significance of each node.

The effects of osmotic stress on wheat seedlings

To analyze the effects of osmotic stress, five different osmotic treatments (0%, 5%, 10%, 15% and 20% PEG-6000) were performed on wheat seedlings at the two-leaf stage. After cultivation for 24 h, the plant height and main root length were severely restrained by osmotic stress, declining to 8.76 and 8.74 cm from 11.88 and 10.13 cm, respectively (Figs. 2A and 2B). The RWC of whole plants was measured, and this value was significantly different between the control and osmotic treatment samples. The RWC was 89.92% after a 0% PEG-6000 treatment and decreased to 81.44% after a 15% PEG-6000 treatment. However, when the PEG-6000 treatment increased to 20% from 15%, the RWC only decreased 0.48%, and this difference was not statistically significant (Fig. 2C). Based on these results, we found that treatment with 10% PEG-6000 for 24 h should be considered as mild osmotic stress (MOS), while treatment with 15% PEG-6000 for 24 h should be considered as severe osmotic stress (SOS).

Identification of root proteins under osmotic stress using iTRAQ

As roots directly sense osmotic stress, total protein was extracted from the root samples of wheat plants under control, MOS and SOS conditions (two, three and three replicates, respectively). The protein expression profiles of these eight root samples were analyzed in one 8-plex iTRAQ experiment. A total of 150,440 triggered MS/MS spectra were identified, and 2,228 proteins were identified by 7,392 peptides (File S2), and about 45.17% of the identified proteins included at least two unique peptides.

The DEPs between osmotic stress and control

The 95% confidence interval of each group distribution was constructed to analyze the maximum scope of difference within group, and the results showed that the maximum scope is 0.855–1.17 (File S2). To insure the difference between groups larger than the difference within group, a fold-change of more than 1.2 (more than 1.2 or less than 0.833) was selected as one of the parameters for DEPs selection. To enhance the confidence of DEPs, the following parameters were also considered: each protein with two or more peptides, at least two times differential expression among three repetition and a significance level of p < 0.05. Based on these four parameters, a total of 81 DEPs were identified, including 34 DEPs between the MOS and control samples and 64 DEPs between the SOS and control samples. Among these DEPs, 17 DEPs were common in the MOS and SOS samples compared with the control samples, 30 DEPs were down-regulated, and 51 DEPs were up-regulated under osmotic stress.

Analysis of the DEPs between osmotic stress and control

Among these DEPs, the molecular function information for 69 DEPs was identified. Many proteins had functions in processes, such as carbohydrate metabolism, protein metabolism, phytohormones responsive etc., and the plant protection system played important roles in the wheat roots response to osmotic stress (Tables 1 and 2).

Plant protection system

Many studies have demonstrated that ROS and cytotoxin would significantly increase under osmotic conditions, which would induce cellular damage in plants. To prevent the damages, plants have generated many plant protection systems to remove ROS and cytotoxins. Here, we found that the proteins involved in ROS scavenging and detoxifying were up-regulated, except some peroxidase.

Glutathione (GSH) has multiple functions, such as antioxidant and detoxification, in plants (Noctor et al., 2012). The ROS would be continuous eliminated by GSH-GSSH (Glutathione disulfide) cycle in organism, which depends on glutathione reductase and glutathione peroxidase. And GSTs, which could be induced through different biotic and abiotic stresses, would protect organisms against oxidative damage and lipid peroxidation, and catalyze the conjugation of electrophilic substrates and glutathione to eliminate cytotoxic substrates (Marrs, 1996; Chen et al., 2012; Yang et al., 2001). In the present study, one glutathione reductase and one glutathione peroxidase were found to be up-regulated (Fig. 3), indicating that GSH-GSSH cycle was more active to maintain ROS balance, under osmotic stress. And eight GSTs were also up-regulated to detoxify harmful materials and maintain cell redox homeostasis in plants under osmotic stress (Fig. 3). In addition, one glutamate-cysteine ligase, which catalyzes the first and rate-limiting step of glutathione biosynthesis, was up-regulated (Fig. 3). All these results showed that glutathione system played important roles in protecting organism from damage caused by osmotic stress in wheat roots.

To better understand the evolutionary relationships of these GSTs, an unrooted phylogenetic tree, including AtGSTs and these eight GSTs, was constructed. We identified two GSTs belonging to the Phi family, two GSTs belonging to the Lambda family and four GSTs belonging to the Tau family (Fig. 4). Most GSTs are Phi or Tau, which are plant-specific GSTs and the major phase II enzymes in a common detoxification pathway (Frova, 2003). Transgenic plants over-expressing Tau or Phi GSTs showed high tolerance to herbicides, salt and UV radiation (Karavangeli et al., 2005; Benekos et al., 2010; Jha, Sharma & Mishra, 2011). These results indicate that glutathione play an important role in the detoxification of cytotoxin under osmotic stress in wheat.

In addition, many other DEPs associated with redox reactions, such as reductase and oxidase, were observed under osmotic stress.