Environmental Pollution 201 (2015) 150e160

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Comparative proteomic analysis in Miscanthus sinensis exposed to antimony stress Liang Xue a, b, 1, Huadong Ren a, 1, Sheng Li a, Ming Gao a, b, Shengqing Shi b, Ermei Chang b, Yuan Wei b, Xiaohua Yao a, Zeping Jiang b, Jianfeng Liu b, * a b

Research Institute of Subtropical Forestry, Chinese Academy Forestry, Fuyang, Zhejiang 311400, China State Key Laboratory of Tree Genetics and Breeding, Research Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 December 2014 Received in revised form 27 February 2015 Accepted 2 March 2015 Available online 21 March 2015

To explore the molecular basis of Sb tolerance mechanism in plant, a comparative proteomic analysis of both roots and leaves in Miscanthus sinensis has been conducted in combination with physiological and biochemical analyses. M. sinensis seedlings were exposed to different doses of Sb, and both roots and leaves were collected after 3 days of treatment. Two-dimensional gel electrophoresis (2-DE) and image analyses found that 29 protein spots showed 1.5-fold change in abundance in leaves and 19 spots in roots, of which 31 were identified by MALDI-TOF-MS and MALDI-TOF-TOF-MS. Proteins involved in antioxidant defense and stress response generally increased their expression all over the Sb treatments. In addition, proteins relative to transcription, signal transduction, energy metabolism and cell division and cell structure showed a variable expression pattern over Sb concentrations. Overall these findings provide new insights into the probable survival mechanisms by which M. sinensis could be adapting to Sb phytotoxicity. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Proteomic Antimony Stress Miscanthus sinensis

1. Introduction Antimony is a genotoxic and carcinogenic element (Huang et al., 1998; Takahashi et al., 2002; Beyersmann and Hartwig, 2008). Sb and its compounds are classified as priority pollutants by the European Union and the Environmental protection Agency of the United States (Johnson, 2008). In China, almost 1,50,000 tons of Sb is produced annually, which account for 70% of world's total output (Information Center of Ministry of Land and Resources of People's Republic of China, 2006). Consequently, much higher concentrations of Sb are found in contaminated environment due to its unreasonable exploitation and utilization (He et al., 2012). A field survey around the Xikuangshan mining area (in Hunan province, China) showed that the concentrations of Sb at the sites were 610e54,221 mg kg
1 (Wei et al., 2011). High levels of Sb in local agricultural soils, crops, and people indicated that the pollutants

* Corresponding author. E-mail address: [email protected] (J. Liu). 1 Liang Xue and Huadong Ren contributed equally to this work. http://dx.doi.org/10.1016/j.envpol.2015.03.004 0269-7491/© 2015 Elsevier Ltd. All rights reserved.

pose a major environmental and human health problem (He et al., 2012; Wu et al., 2011). Phytoremediation is using the plants and their associated rhizospheric microorganisms to remove, degrade, or immobilize various pollutants from contaminated sites (Tong et al., 2004). It has gained acceptance as a cost-effective and ecological-effective technology to ameliorate contaminated sites (Salt et al., 1998). Both technologies, phytoextraction and phytostabilization require metal tolerant plant species. Nevertheless, the lack of ideal plants with sufficient biomass and high growth rates inhibits the development of technology for Sb remediation. To overcome the limitations, development of genetically modified plants with the desired characteristics mentioned above is largely in demand. Therefore, a better understanding of physiological and molecular regulation mechanisms of accumulating and detoxificating Sb become essential. In general, physiological changes such as inhibition of growth and development, reduction in photosynthesis, disturbance of redox control activity and nutrient element absorption occur under Sb stress (Tschan et al., 2009; He and Yang, 1999; Feng et al., 2009; Pan et al., 2011; Zhang et al., 2012; Shtangeeva et al., 2011; Paoli et al., 2013). However, the molecular mechanism of excess Sb tolerance is poorly understood. In

L. Xue et al. / Environmental Pollution 201 (2015) 150e160

plants, only Sb-binding polypeptides and Sb-transport proteins have been identified and characterized (Maeda et al., 1998; Wysocki et al., 2003; Kamiya and Fujiwara, 2009). Miscanthus sinensis, a perennial rhizomatous, fast growing high biomass grass, accumulates higher amount of Sb at extremely toxic levels (Lewandowski et al., 1995; Xue et al., 2014). These suggest that M. sinensis is an ideal candidate for phytoremediation of Sb contaminated sites. Proteomic analysis of M. sinensis under Sb stress could provide a more in-depth understanding on the Sbtolerant mechanism of plants at molecular level, in light of a large amount of plant proteins change in the types and quantities under abiotic stress (Ahsan et al., 2009). In the present study, we analyzed the variations of total proteins in M. sinensis roots and leaves after Sb treatment with the following objectives: (1) to identify proteins potentially involved in Sb tolerance, Sb translocation, Sb accumulation, or the regulation of Sb responses in M. sinensis; (2) to explore possible Sb tolerance/ accumulation mechanism existing in M. sinensis; and (3) to contribute to establishing reference data sets on plant proteome changes to Sb stress. 2. Materials and methods 2.1. Plant growth and treatments M. sinensis seeds were collected from plants growing on Sb-mining spoil deposits in Lengshuijiang County of Hunan province of china. Seeds were surface sterilized with 70% (v/v) ethanol and 0.5% (v/v) sodium hypochlorite prior to imbibing for 16 h in distilled water with aeration. Seeds were then transferred to commercial potting mix in plastic trays and allowed to germinate in a growth chamber. After 28 days of growth, the uniform seedlings were transferred to hydroponic cultures supplied with half strength Hoagland nutrient solution. The pH was adjusted to 5.8 with HCl. Following a 1-week hydroponic adaptation, the seedlings were subjected to new nutrient solutions for another 3 days treatments of 0, 50, 100, 200, 300, 500, 750 and 1000 mm KSb(OH)6. The entire experiment was conducted under light  conditions (500 mmol m
2 s
1, 16/8 h light/dark period) at 25 C and 65% humidity. Roots and leaves were cut off from the Sb-treated plant, pooled, rinsed quickly in deionized water, quickly frozen by liquid nitrogen, and stored at
80  C for protein extraction. Other parts of roots and leaves were used to determine the tissue Sb concentrations. 2.2. Determination of Sb accumulation After 3 days of treatment, plant samples were divided into two parts (root and shoot) and washed with deionized water for approximately 3 min to remove surface Sb salt. Samples were dried at 80  C for 2 days and then ground to a fine powder using a Wiley Mill (Thomas Model 4, USA). About 0.2 g of plant powder were mineralized with H2O2/HF/HNO3 at a ratio of 2:5:10 (U.S. Environmental Protection Agency, 1996). Digestion of samples was carried out in a microwave digestion system (Milestone Ethos 1, Italy). Sb content in the digested solution was determined by fluorescence spectrophotometry (F-4500; Hitachi, Japan). Certified reference materials (GBW07604, China) was utilized to assure the analytical quality. The translocation factor (TF) indicates the efficiency of a plant in translocating Sb from root to leaf and is calculated as follows:

151

2.3. Measurement of physiological response Leaf content of M. sinensis was measured by portable chlorophyll meter (SPAD-502, Konicam, Japan). Lipid peroxidation in M. sinensis was estimated by determining the concentration of the malonaldehyde (MDA). MDA content was extracted and measured by thiobarbituric acid (TBA) reaction according to Kosugi and Kikugawa (1985). Absorbance of supernatant was monitored at 450 nm, 532 nm and 600 nm, respectively. The lipid peroxidation concentration was expressed as mmol g
1 fresh weight. To determine free proline level, 0.5 g of leaf samples were homogenized in 5 ml 3% (w/v) sulfosalicylic acid and heated at 100  C for 10 min. Reaction was stopped by ice bath, then 2 ml supernatant was extracted and added by 2 ml glacial acetic acid and 3 ml color reagent. After heated at 100  C for 40 min, the mixture was extracted with toluene and the absorbance of fraction with toluene was read at 520 nm. Proline level was determined using calibration curve and expressed as mg$g
1 fresh weight. 2.4. Protein extraction and 2-D electrophoresis For each sample extraction, 1 g of M. sinensis roots or leaves was ground with liquid nitrogen using sterile pestle and mortar. Then each of tissue samples was resuspended in 3 ml of ice-cold extraction buffer (50 mmol L
1 TriseHCl, pH 8.5; 5 mmol L
1 EDTA; 100 mmol L
1 KCl; 1% DTT; and 30% sucrose) and fractionated with water-saturated phenol, followed by centrifugation at 6000 g for 3 min. The proteins were recovered from the supernatant by precipitation with 0.1 mol L
1 ammonium acetate in methanol. The protein pellets were washed twice with a cold solution of 80% acetone. After air-dried the proteins were resuspended in 2-DE solubilization buffer (40 mM Tris, 7 M Urea, 2 M Thiourea, 1 mM EDTA$Na2, 4% CHAPS, 1% DTT). The concentration was quantified using the Bradford method (Bradford, 1976). Approximately 1 mg dissolved protein sample was applied to a 24 cm, pH 4e7 immobilized gradient IPG (Bio-Rad). After isoelectric focusing (IEF) for 1,00,000 V h, the strips were equilibrated for 15 min in equilibration solution A (6 M urea, 0.375 M TriseHCl, pH 8.8, 2% SDS, 20% glycerol, 2% DTT) and in equilibration solution B (6 M urea, 0.375 M TriseHCl, pH 8.8, 2% SDS, 20% glycerol, 2.5% iodoacetamide) for another 15 min. Second dimension electrophoresis was carried out on a 13% SDS-PAGE gel containing 30%/0.8% Acr/Bis, 1.5 M Tris, pH 8.8, 10% SDS, 10% APS and trace TEMED using an Ettan DALT Twelve System (Amersham bioscience) (Laemmli, 1970). The electrophoresis was carried out at 25  C and 5 mA per gel until the samples departed from the strips and then 25 mA per gel until the bromophenol blue dye front arrived at the bottom of the gels. Following the SDS-PAGE, electrophoresed proteins were stained with Coomassie R-350 (GE Healthcare). 2.5. Gel visualization and image analysis The gels were scanned by a UMAX Powerlook 2100XL scanner (Maxium Technologies, Taipei, China) and analyzed using the Image Master™ 2D Platinum software version 7.0 (Amersham Biosciences, Sweden). Relative comparison of the intensity abundance among the samples was performed using KolmogoroveSmirnov test. The protein spots with distinct differences were regarded to have at least a 1.5-fold amount of change.

TF ¼ Cleaf/Croot

2.6. Protein identification

where Cleaf and Croot are the concentrations (mg kg
1) of Sb in the plant leaf and root, respectively.

Selected protein spots were excised manually from the CBBstained gels, washed with destaining solution (ACN: 0.1 M

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NH4HCO3 ¼ 3:7). After washing, the gel spots were dehydrated with 100% ACN for 5 min and then vacuum-dried for 15 min. The protein spots were then digested by 10 ml trypsin for 30 min at 4  C, followed by an addition of 10 ml solution containing 5% ACN and 25 mM NH4HCO3 and with an incubation overnight at 37  C. The supernatant was extracted three times in 33% CAN contented 0.1% TFA. The vacuum-dried peptides were subjected to mass spectroscopy using an Autoflex II MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Germany). The peptide mass fingerprint (PMF) and MS/MS data were used to derive the protein identity using the MASCOT search engine (http://www.matrixscience.com) applied to the NCBInr 20120908 release (20191133 sequences; 6915215180 residues). Proteins were successfully identified based on 95% or higher confidence interval of their scores in search engine. The search parameters were NCBInr green plants database, trypsin as the digestion enzyme, one missed cleavage site, fixed modifications of Carbamidomethyl (C), variable modifications of Oxidation (M), 150 ppm for peptides tolerance and 0.6 Da for TOFeTOF fragments tolerance, and the MHþ and monoisotopic for mass values. BLASTP (http://www.ncbi.nlm.nih.gov/BLAST/) was used to search for homologs of the unknown proteins.

2.7. Statistical analysis Results of the physiological parameters and spot intensity were statistically analyzed by using analysis of variance (ANOVA) and Duncan's multiple range test (DMRT) to determine significant differences among group means. Significant differences from control values were determined at p < 0.05 levels. All the results are represented as means ± SE of at least three independent replicates. The statistical package SPSS version 17.0 (SPSS inc., Chicago, USA) was used for the statistical analyses.

2.8. Hierarchical cluster analyses The expression profiles of differential proteins were analyzed through a hierarchical clustering according to the cluster version 3.0 which was available from a website (http://rana.lbl.gov/ EisenSoftware.htm). Rows were mean centered, and Euclidean distance and Average Linkage were used for data aggregation. The dendrogram was visualized using Java TreeView version 1.1.3, which was also available from a website (http://sourceforge.net/ projects/jtreeview.htm). 2.9. Gene ontology annotation The gene ontology (GO) database (http://geneontology.org/) was used to classify proteins on the basis of biological function. The GO annotations and numbers were retrieved and were grouped into different levels, and pie charts were generated. 3. Results 3.1. Morphological and physiological symptoms of Sb treatment After exposure over 500 mM Sb for 3 days, the plants showed evident symptoms of growth suppressed in leaves and roots (Fig. 1A). However, MDA content in leaves significantly changed from 750 mM of Sb with the highest increase by 121.03% at 1000 mM (Fig. 1B). Chlorophyll content showed a constant decrease after antimony stress (Fig. 1C). Analysis of POD indicated significant changes in activity upon antimony treatment. The POD activity peaked in response to 1000 mM. Furthermore, the POD levels were significantly higher than the control value under all three treatments, reaching up to 256% at 1000 mM Sb (Fig. 1D). Comparing to control, the analysis of proline content in leaves showed that the

Fig. 1. Effect of Sb on the growth, MDA, chlorophyll and POD in M. sinensis. Values (mean ± SD) were determined from three independent experiments (n ¼ 3) after plants had been treated with 0, 500, 750 and 1000 mM Sb for 3 d.

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153

Fig. 2. Effect of Sb on content of proline and concentration of Sb in M. sinensis. Values (mean ± SD) were determined from three independent experiments (n ¼ 3) after plants had been treated with 0, 500, 750 and 1000 mM Sb for 3 d.

amount of proline increased after Sb stress (Fig. 2A). Sb accumulation in M. sinensis roots and leaves increased with rising concentrations of KSb(OH)6. After 3 days of exposure to 1000 mM, the root and leaf tissues accumulated 163 mg/kg and 29 mg/kg Sb on a dry wet, respectively (Fig. 2B). At 1000 mM Sb, M. sinensis peaked the highest TF value (0.18). This pattern of accumulation agreed

with former reports that roots of plants accumulate much higher contents of Sb than do the leaves. 3.2. Protein expression profiles Proteins extracted from M. sinensis leaves and roots following Sb

Fig. 3. Representative 2-DE gel images of M. sinensis leaves and roots treated with Sb. (A) Identified proteins showing significant (p < 0.05) changes in leaves. (B) Identified proteins showing significant (p < 0.05) changes in roots. (C) Close-up views of differentially expressed protein spots in M. sinensis (highlighted by circle).

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Fig. 4. Relative expression levels of differentially expressed proteins in M. sinensis leaves and roots under Sb stress. Three treatments including control (500, 750, 1000 mM Sb for 3 d) were performed. Different letters above the bars indicate statistically significant differences (p < 0.05).

treatment were separated by 2-DE. Using the Image Master software, more than 1300 protein spots were reproducibly detected in M. sinensis leaves and 580 spots in roots, respectively (Fig. 3). 29 protein spots showed 1.5-fold change in abundance in leaves and 19 spots in roots (Figs. 3 and 4). Average expression of these protein spots were compared across the three treatments. The relative abundance of 31 successfully identified protein spots on the gel is

shown in Fig. 4. 3.3. Protein identification and pathway analysis The main protein spots showing significant differences between Sb treatment and controls were selected for MS or MS/MS analysis. The differentially expressed proteins in M. sinensis roots and leaves

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155

Table 1 List of protein identified by mass spectrometry in M. sinensis leaf and root. Spot no.

NCBI accession

Protein name

Biological function

Method

(A) M. sinensis leaf proteins with at least 1.5-fold change in abundance after 500 mM, 750 mM and 1000 mM Sb treatment 11 gij224070969 Predicted protein Unclassified PMF 49 gij56675682 ATP synthase beta subunit Energy metabolism PMF 70 gij365189292 Heat shock protein 90 Stress response PMF 98 gij15238798 protein-serine/threonine kinase Signal Transduction PMF 168 gij77552489 Hypothetical protein LOC_Os11g45640 Unclassified PMF 183 gij297801228 Hypothetical protein ARALYDRAFT_330262 Unclassified PMF 195 gij225423424 alpha-1,4-glucan-protein synthase Energy metabolism PMF 221 gij11558242 Peroxiredoxin Antioxidant defense MS/MS 229 gij17064850 Protein kinase 5 Signal Transduction PMF 233 gij302766982 Hypothetical protein SELMODRAFT_408167 Unknown PMF 247 gij381147553 Maturase K Transcription PMF 285 gij357115243 Peroxidase 73-like Antioxidant defense MS/MS 316 gij559005 Ascorbate peroxidase Antioxidant defense MS/MS 346 gij5923877 Glutathione S-transferase Antioxidant defense MS/MS 551 gij401715664 Predicted protein Unknown PMF 559 gij62732954 Fructose- bisphosphate aldolase I Signal Transduction MS/MS 999 gij297612744 Os12g0169400 Unknown PMF 1350 gij255103505 RuBisCOLSU Photosynthesis PMF 1443 gij4678941 Gamma response I protein Cell division and cell structure PMF 1698 gij359481615 PREDICTED: sorting nexin-1 Unclassified PMF B) M. sinensis root proteins with at least 1.5-fold change in abundance after 500 mM, 750 mM and 1000 mM Sb treatment 30 gij307110760 Hypothetical protein CHLNCDRAFT_29444 Unknown PMF 31 gij58978027 Pathogenesis-related protein 10d Stress response MS/MS 53 gij222424107 AT5G27830 Antioxidant defense PMF 58 gij384249944 C-signal Antioxidant defense PMF 61 gij381147553 Maturase K Transcription PMF 62 gij242050276 Hypothetical protein SORBIDRAFT_02g033760 Unknown MS/MS 65 gij357147625 PREDICTED: golgin candidate 4-like Cell division and cell structure PMF 96 gij559005 Ascorbate peroxidase Antioxidant defense MS/MS 121 gij5923877 Glutathione S-transferase Antioxidant defense MS/MS 204 gij42568723 jumonji (jmjC) domain-containing protein Transcription PMF 278 gij4388533 F1-ATP synthase, beta subunit Energy metabolism PMF a b c d

TheoMr (kDa)/pIa

Scoreb

PMc

SC (%)d

12,356/5.18 53,660/5.03 93,283/4.88 65,314/8.96 19,222/5.58 15,449/9.06 41,488/5.66 28,776/5.17 65,313/9.06 39,479/5.57 19,383/7.96 38,549/8.72 27,656/5.43 23,609/5.79 18,326/5.95 39,556/6.0.85 58,552/6.50 22,042/5.5 125,536/7.54 46,094/8.21

66 63 56 52 55 57 52 33 61 64 71 36 19 101 56 69 63 96 61 74

3 4 7 4 3 3 3 1 6 5 5 1 1 1 4 2 4 7 7 6

21 13 12 9 31 33 16 8 11 21 33 9 5 9 34 5 14 35 6 17

48,009/6.72 17,059/5.19 31,562/8.95 27,364/5.44 19383/7.96 18,910/6.51 58,825/5.55 27,656/5.43 23,609/5.79 54,836/4.91 49,219/5.25

60 30 64 57 51 61 62 30 32 51 177

6 1 5 4 3 1 6 1 1 5 14

21 13 30 20 20 15 17 5 9 16 37

Theoretical pI and MW (kDa) of the identified proteins. Mascot score obtained after searching against the NCBI nr database. Match peptides. Amino acid sequence coverage for the identified proteins.

were summarized in Tables 1 and 2, respectively. Among the identified proteins, two enzymes involved in ROS scavenging, were simultaneously up-regulated in both M. sinensis roots (Spot 96， 121) and leaves (Spot 316, 346, 551) at all three Sb concentrations. In addition, another three ROS-related proteins (Spot 58, 221, 285) were up-regulated either in M. sinensis leaves or roots. Moreover, a serine/threonine-protein kinases (Spot 229), a zinc-finger protein (Spot 62), two ATP synthases (Spot 49, 278) and 4 stress proteins including Hsp90 (Spot 70), Dnaj-like protein (spot 30), pathogenesis-related protein 10 (Spot 31) and CBS Domain Containing Protein (Spot 999) also significantly up-regulated under all levels of Sb treatment. In contrast, several proteins such as RuBisCO LSU (Spot 1350), AT5G27830 (Spot 53) and hypothetical proteins (Spot 168, 183) were all down-regulated under any concentration of Sb stress. Remarkably, the expression of maturase K in leaves (Spot 247) was up-regulated under any Sb levels, while a downregulation was observed in its roots (Spot 61). Furthermore, gamma response protein (Spot 1443) hardly detectable in the control sample was induced after treatment, while sorting nexin-1 (Spot 1698) almost disappeared when exposed to Sb. The rest of the proteins mainly possessed fluctuating characteristics among three antimony levels. Gene ontology (GO) annotation and the biological functions allowed to classify the identified proteins into 8 categories viz., Antioxidant defence, photosynthesis, energy metabolism, signal transduction, cell division and cell structure, transcription, stress response and unclassified (Fig. 5). 3.4. Hierarchical clustering analysis The expression profiles of differential proteins were analyzed

through hierarchical clustering method. Rows were mean centered; Euclidean distance and Average Linkage were used for data aggregation. All expression patterns were constructed by a dendrogram, the branch length of which reflects the degree of similarity (Fig. 6A). K-mean cluster analysis was applied to classify the proteins with different expression pattern under Sb stress into 8 clusters. K-mean clustering was also used to categorize the differentially expressed proteins and showed a more clear abundance relationship with Sb treatment. 31 Sbresponsive proteins were categorized in 8 expression groups (Fig. 6B). 4. Discussion In this study, we found that both M. sinensis leaves and roots contained Sb concentration considerably higher than this phytotoxic level and showed no toxic symptom until at 500 mM solution of Sb. Combined with its low TFs, M. sinensis could be used as a potential material for phytostabilization of Sb. The elevated MDA content in M. sinensis after Sb treatment indicated that lipid peroxidation is a kind of Sb phytotoxicity, which were also found in other plants (Feng et al., 2009; Pan et al., 2011; Paoli et al., 2013; Feng et al., 2013a). The chlorophyll synthesis in M. sinensis was inhibited by Sb, which was found to effect plant photosynthesis (Pan et al., 2011). POD is a kind of antioxidants for H2O2-scavenging in plants (Foyer and Noctor, 2000). In the present study, both the activities and relative expression levels of POD increased significantly over all three Sb concentrations, which may play an important role in regulating ROS metabolism. Proline accumulation has been proposed to play a vital role in maintaining osmotic

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Table 2 The homologs of unknown proteins. Spot no.

NCBI accessiona

Homolog protein

Biological function

NCBI accessionb

Coverage

Identities

Expect

30 62 233 551 999

gij307110760 gij242050276 gij302766982 gij401715664 gij297612744

DnaJ-like protein Zac, putative 1-aminocyclopropane-1-carboxylate oxidase L-ascorbate peroxidase CBS domain containing protein

Stress response Transcription Signal Transduction Antioxidant defense Stress response

gij384247665 gij49391105 gij74919899 gij308806560 gij226501428

92 99 82 100 92

61 84 34 44 86

5e-165 3e-100 1e-44 1e-42 0.0

a b

The accession number of the unknown proteins in Table 1. The accession number of the homologs identified by BLAST.

balance, protecting membrane structure, regulating redox status, or scavenging the ROS (Hayat et al., 2012; Sharma and Dietz, 2006). The cluster analysis was done for the 31 Sb-responsive proteins. The data were taken in terms of efold expression with respect to the control value. Then, the data sets were log-transformed to the base 2 to normalize the scale of expression clusters, and only the cluster with n > 5 were used to study the co-expression patterns for functionally similar proteins (Fig. 6B). The largest number of group, cluster 2 proteins showed induction following Sb treatment and were up-regulated under all stress levels (Fig. 6B). This group was found to be enriched in proteins involved in antioxidant enzymes and stress responsive proteins (Fig. 6C). Proteins involved in transcription displayed a diverse and complex pattern of regulation (Cluster 1, 2 and 7) (Fig. 6C). Overall, these up-regulated proteins by Sb stresses suggest future studies on the improvement of Sb tolerance in M. sinensis. 4.1. Antioxidant defense Reactive oxygen species (ROS) induced by excess accumulation of Sb in plants has been reported (Feng et al., 2013b). More than 90% of the proteomic research showed that proteins involving in antioxidative defense are differentially regulated in response to metal stress (Ahsan et al., 2009). A total of 9 ROS-related proteins identified on the proteome map of M. sinensis under Sb stress were classified mainly in Peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX) and Glutathione S-transferase (GST). Among the antioxidant proteins, APXs provide the first line of defense against ROS. APXs possess a function in decomposing H2O2 into water by using ascorbic acid as a specific electron donor (Asada, 1992; Foyer et al., 1994). PODs are ubiquitous in vascular plants, and can degrade H2O2 by oxidation of co-substrates (Blikhina et al., 2003). It has been suggest that the enhanced activities of APX and POD are important tolerance mechanisms in Sbtolerant plants (Feng et al., 2009). Prxs act as thiol-dependent POD

and have the primary function in protecting other targets from oxidation (Dietz et al., 2006). Thus, the up-regulation of APXs (Spot 96，316, 551), POD (Spot 285) and Prx (Spot 221) under Sb stress in M. sinensis are reasonable in the light of previous research. GSTs are multifunctional proteins involved in the cellular detoxification (Jain et al., 2006). In addition, differential expression of proteins of the GST family has been observed in almost all proteome research related to metal toxicity (Roth et al., 2006; Ahsan et al., 2008; Smith et al., 2004). The up-regulation of GST (Spot 121, 346) under Sb exposure in M. sinensis were consistent with multiple studies involving proteomic responses related to metal stress, all of which identify GSTs as second-line antioxidant defense system under metal exposure. Lastly, compared with control M. sinensis the Sb treated samples exhibited an up-regulation of C-signal (Spot 58) and a downregulation of a putative ROS-related protein AT5G27830 (Spot 53). Since the functions of these proteins are not clearly known, a possible role in regulating ROS can only be speculated in M. sinensis under Sb stress. 4.2. Photosynthesis Plants can accumulate metals in its leaves by transporter and other mechanism when exposed to metals. The accumulation of metals in leaves can severely impact on photosynthetic pathway through reducing the chlorophyll content and decreasing the net photosynthesis (Führs et al., 2008; Bona et al., 2007; Kieffer et al., 2008). A proteomic analysis of rice leaves showed that 17 proteins involved in photosynthetic pathways were differentially expressed in comparison with the control by exposure to a variety of metals including Cu, Zn, Cd, Hg, Co, Li and Sr (Hajduch et al., 2001). Duquesnoy et al. showed that four large and small subunits of RuBisCO (RuBisCO LSU and SSU) involved in primary carbon metabolism and photosynthesis disappeared in Agrostis tenuis leaves compared with the control (Duquesnoy et al., 2009). Combined with the reduction in chlorophyll content, the large subunits of RuBisCO (RuBisCO LSU) (Spot 1350) were decreased in abundance under Sb stress, suggesting that Sb disrupts the photosynthetic machinery in M. sinensis. 4.3. Energy metabolism

Fig. 5. Pie chart illustrating the assignment of the identified proteins to functional categories.

Several proteins involved in energy metabolism were identified in this study. These include ATP synthase beta subunit (Spot 49), F1ATP synthase beta subunit (Spot 278) and Alpha-1,4-glucan-protein synthase (Spot 195). In many proteomic analysis on the effects of toxic metals on plants, energy metabolism is disturbed (Castillejo et al., 2012; Gillet et al., 2006; Lee et al., 2010). To maintain normal growth, amount of defense mechanisms call for activation, all of which require energy. Acceleration of energy-dependent reactions is apparent from the higher expression of ATP synthase (Spot 49, 278), which allows releasing energy from ATP. The enzyme alpha-1,4-glucan-protein

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157

Fig. 6. Clustering analysis of the differentially expressed proteins of M. sinensis under Sb stress. (A) Dendrogram of the spots clustering is showed in the top. (B) K-means clustering showing the expression patterns for individual protein spots in the eight main Sb-responsive clusters are shown below. The expression profile of each individual protein in the cluster is depicted by blue lines while the mean expression profile is marked in pink for each cluster. (C) Pie chart illustrating the Sb-responsive clusters (n > 5) to functional categories. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

synthase acting as a glycosyltransferase, is involved in the cell wall polysaccharides (Castillejo et al., 2012). Recent investigations have shown an up-regulation of this enzyme in plants exposed to diverse

abiotic stresses (Castillejo et al., 2012; Gillet et al., 2006; Lee et al., 2010). Intriguingly, this protein (Spot 195) in M. sinensis was upregulated at 750 mM Sb treatment and returned to normal

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expression at 1000 mM compared with the controls, which indicated that excess Sb addition may inhibit its activity. 4.4. Signal transduction Plants adopt various mechanisms when exposed to abiotic environmental conditions. Combined with proteomic, metabolomic and biochemical analyses, it was proved that there is a strong correlation between metal stress and the expression of signaling molecules in plants (Maksymiec et al., 2005; RodríguezSerrano et al., 2006). In this study, four proteins (Spot 98, 229, 233, 559) involved in signaling pathways were observed. We found that the level of ACC oxidase (Spot 233) in M. sinensis increased during Sb stress. ACC oxidase catalyses the final step of ethylene synthesis using ACC as substrate. Ethylene acts as a plant hormone involving not only in numerous aspects of growth and development, but also in stress signaling (Kieber, 1997). Wi and Park (2002) showed that antisense expression of ethylene biosynthesis genes ACC oxidase enhanced a broad spectrum of abiotic stress tolerance in transgenic tobacco plants. Our result suggested that the ethylene signaling pathway is involved in the response to Sb exposure. It has been documented that serine/threonine-protein kinase possesses a function in catalyzes the phosphorylation of serine or threonine residues on target proteins, by which the function of the target protein may be changed (Hardie, 1999). The present study showed two serine/threonine protein kinases (Spot 98, 229) differentially expressed under Sb stress compared with the controls, which may play an important role in signaling events. The expression patterns of Fructose 1, 6-biphosphate aldolase (FBA) in Arabidopsis under different stress conditions suggested that all the members showed different expression patterns in response to diverse abiotic stresses, including ABA, NaCl, Cd, abnormal temperature and drought (Lu et al., 2012). A homologous FBA protein (Spot 559) was differentially expressed when exposed to Sb treatment, which supported the previous results. 4.5. Cell division and cell structure Gamma response 1 protein (GR1) is a key enzyme in mediating cell cycle arrest before mitosis in response to DNA damage. It has been demonstrated that a putative transcription factor governing multiple responses to DNA damage was encoded by suppressor of GR1 (Yoshiyama et al., 2009). Intriguingly, GR1 (Spot 1443) in leaf tissues of M. sinensis was markedly induced by Sb stress. This result suggested that the cell division of plants were highly affected by Sb stress and the induction of GR1 possibly mediate the cell cycle arrest to reduce the genotoxicity of Sb. In addition, a putative protein golgin candidate 4 (Spot 65) was down-regulated under Sb treatment, which play a significant role in fixing Golgi membrane vesicles and maintaining the structure of Golgi apparatus. 4.6. Transcription Three differential proteins were involved in transcription. Maturase K is a kind of maturase participating in catalyzing intron RNA binding and spicing as well as directly regulating gene expression at transcriptional level (Ji et al., 2009). Maturase K in response to abiotic stress (e.g. acid rain, salt, ROS, drought) has been widely reported (Hu et al., 2014; Pandey et al., 2008; Zorb et al., 2010). In this study, maturase K (Spot 247) was up-regulated in M. sinensis leaves but was down-regulated in M. sinensis roots (Spot 61) during Sb stress. The zinc finger proteins (ZFP) are a super family of proteins involved in regulating resistance mechanism for various biotic and abiotic stresses (Feurtado et al., 2011; Giri et al.,

2011). Overexpression of a ZFP gene OSISAP1 from rice enhanced tolerance to cold, dehydration and salt stress in transgenic tobacco (Mukhopadhyay et al., 2004). The present research showed that the abundance of a putative ZFP protein Zac (Spot 62) in M. sinensis roots was increased when exposed to Sb, indicating that the upregulation of Zac may play an important role in enhancing Sb tolerance in M. sinensis. In addition, transcription factor jumonji (jmjC) domain-containing protein (Spot 204) was also differently expressed with Sb treatment, which may possess the enzymatic activity (Klose et al., 2006). 4.7. Stress response A group of proteins known as molecular chaperones assist the protein folding, assembly, degradation and translocation. Heat shock proteins HSPs are chaperones playing a crucial role in plant protection against stress by rebuilding normal protein conformation. Hsp90 (Spot 70) was up-regulated under all sb stress conditions. HSP90 mediates various cellular processes including cell cycle control, cell survival, hormone signaling and stress response, functioning as a key component in maintaining cellular homeostasis (Wandinger et al., 2008; Zhao et al., 2005). In addition, a Dnaj-like (Spot 30) was also up-regulated under all Sb stress conditions. Dnaj proteins act as co-chaperones in protein homeostasis and protein complex stabilization under adverse conditions (Kong et al., 2014). Zhang et al. (2000) showed that Dnaj-like proteincoding genes in Phaseolus vulgaris was up-regulated under Hg and Cd treatment, indicating that DnaJ-like protein may play a significant effect in maintaining cell structure and function under heavy metal stress. Together with these results, it was supposed that these chaperones play an important role in plant-acquired Sb stress tolerance. Pathogenesis-related protein (PR) serves as an important part of plant defense system induced under such pathological related environment. PR-10 is a class of PR family functioning as nucleic acid enzyme. Hajduch et al. (2001) showed that PR-10 in rice were induced by many species of metal such as Cu, Cd and Hg. Kim made a further research on the feature of PR-10 in rice, and find it was related to aging process and had nucleic acid enzymatic activity (Kim et al., 2008). CBS Domain Containing Protein was implicated in enhancing plant tolerance to variety of abiotic stress such as salt, oxidation and heavy metals (Singh et al., 2012). In this study, PR10d (Spot 31) and CBS domain-containing Protein (Spot 999) were all up-regulated by exposure to Sb, suggesting that the proteins play a crucial role in resisting to external stresses on M. sinensis. 4.8. Unclassified Suppression in sorting nexin-1 (Spot 1698) in response to Sb was detected, which led to detrimental effects on cellular communication, intracellular proteins transportation and Protein catabolism. Furthermore, three unknown putative proteins predicted protein (Spot 11), hypothetical protein LOC_Os11g45640 (Spot 168), hypothetical protein ARALYDRAFT_330262 (Spot 183) were also detected under Sb treatment, the functions of these proteins involved in Sb response need a further study. 5. Conclusions In summary, the analysis of physiological, biochemical proteomic responses was conducted in this work provides new insights to the effect of Sb on M. sinensis. A total of 31 differentially expressed proteins were identified which are functionally involved in antioxidant defence, photosynthesis, energy metabolism, signal

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transduction, cell division and cell structure, transcription, stress response and unclassified. Candidates conferring Sb tolerance include Dnaj-like protein, CBS domain-containing protein, HSP90, PR, ZFP, GR1, ACC oxidase, ATP synthase, APX, POD, Prx and GST. The majority of these proteins were involved in antioxidant defense and stress response, which may play a central role in the detoxification of Sb stress. The genes encoding these differentially expression proteins should be further investigated, which may enable us to develop strategies for efficient Sb phytoremediation in fast growing high biomass plants. Acknowledgments The work was funded by the Ministry of Science and Technology, China (2012BAC09B03 and 2011BAD38B0103). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2015.03.004. References Ahsan, N., Lee, D.G., Alam, I., Kim, P.J., Lee, J.J., Ahn, Y.O., Kwak, S.S., Lee, I.J., Bahk, J.D., Kang, K.Y., Renaut, J., Komatsu, S., Lee, B.H., 2008. Comparative proteomic study of arsenic-induced differentially expressed proteins in rice roots reveals glutathione plays a central role during as stress. Proteomics 8, 3561e3576. Ahsan, N., Renaut, J., Komatsu, S., 2009. Recent developments in the application of proteomics to the analysis of plant responses to heavy metals. Proteomics 9, 2602e2621. Asada, K., 1992. Ascorbate peroxidase e a hydrogen peroxide scavenging enzyme in plants. Physiol. Plant 85, 235e241. Beyersmann, D., Hartwig, A., 2008. Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms. Arch. Toxicol. 82, 493e512. Blikhina, O., Virolainen, E., Fagerstedt, K.V., 2003. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. 91, 179e194. Bona, E., Marsano, F., Cavaletto, M., Berta, G., 2007. Proteomic characterization of copper stress response in Cannabis sativa roots. Proteomics 7, 1121e1130. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254.  Ferna ndez-Aparicio, M., Rubiales, D., 2012. Proteomic analysis by Castillejo, M.A., two-dimensional differential in gel electrophoresis (2D DIGE) of the early response of Pisum sativum to Orobanche crenata. J. Exp. Bot. 63, 107e119. Dietz, K.J., Jacob, S., Oelze, M.L., Laxa, M., Tognetti, V., de Miranda, S.M., Baier, M., Finkemeier, I., 2006. The function of peroxiredoxins in plant organelle redox metabolism. J. Exp. Bot. 57, 1697e1709. Duquesnoy, I., Goupil, P., Nadaud, I., Branlard, G., Piquet-Pissaloux, A., Ledoigt, G., 2009. Identification of Agrostis tenuis leaf proteins in response to As(V) and As(III) induced stress using a proteomics approach. Plant Sci. 176, 206e213. Feng, R.W., Wei, C., Tu, S., Wu, F., Yang, L., 2009. Antimony accumulation and antioxidative responses in four fern plants. Plant Soil 317, 93e101. Feng, R.W., Wei, C.Y., Tu, S.X., Liu, Z.Q., 2013. Interactive effects of selenium and antimony on the uptake of selenium, antimony and essential elements in paddy-rice. Plant Soil 365, 375e386. Feng, R.W., Wei, C.Y., Tu, S.X., 2013. The roles of selenium in protecting plants against abiotic stresses. Environ. Exp. Bot. 87, 58e68. Feurtado, J.A., Huang, D., Wicki-Stordeur, L., Hemstock, L.E., Potentier, M.S., Tsang, E.W., Cutler, A.J., 2011. The Arabidopsis C2H2 zinc finger indeterminate domain1/enhydrous promotes the transition to germination by regulating light and hormonal signaling during seed maturation. Plant Cell. 23, 1772e1794. Foyer, C.H., Noctor, G., 2000. Oxygen processing in photosynthesis: regulation and signaling. New. Phytol. 146, 359e388. res, P., Kunert, K.J., 1994. Protection against oxygen radicals: Foyer, C.H., Descourvie an important defence mechanism studied in transgenic plants. Plant Cell. Environ. 17, 507e523. Führs, H., Hartwig, M., Molina, L.E., Heintz, D., Van Dorsselaer, A., Braun, H.P., Horst, W.J., 2008. Early manganese-toxicity response in Vigna unguiculata L. ea proteomic and transcriptomic study. Proteomics 8, 149e159. chal, P., 2006. Cadmium response Gillet, S., Decottignies, P., Chardonnet, S., Le Mare and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach. Photosynth. Res. 89, 201e211. Giri, J., Vij, S., Dansana, P.K., Tyagi, A.K., 2011. Rice A20/AN1 zinc-finger containing stress-associated proteins (SAP1/11) and a receptor-like cytoplasmic kinase (OsRLCK253) interact via A20 zinc-finger and confer abiotic stress tolerance in transgenic Arabidopsis plants. New. Phytol. 191, 721e732. Hajduch, M., Rakwal, R., Agrawal, G.K., Yonekura, M., Pretova, A., 2001. High-resolution two-dimensional electrophoresis separation of proteins from metal-

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