Evaluation of proteome alterations induced by cadmium stress in sunflower (Helianthus annuus L.) cultures

Ecotoxicology and Environmental Safety 119 (2015) 170–177

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Evaluation of proteome alterations induced by cadmium stress in sunflower (Helianthus annuus L.) cultures Cícero Alves Lopes Júnior a,b, Herbert de Sousa Barbosa a,b,c, Rodrigo Moretto Galazzi a,b, Hector Henrique Ferreira Koolen b,d, Fábio Cesar Gozzo b,d, Marco Aurélio Zezzi Arruda a,b,n a Spectrometry, Sample Preparation and Mechanization Group – GEPAM, Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil b National Institute of Science and Technology for Bioanalytics, Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil c Department of Chemistry, Federal University of Piauí – UFPI, P.O. Box 6154, 64049-550 Teresina, PI, Brazil d Dalton Mass Spectrometry Group, Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil

art ic l e i nf o

a b s t r a c t

Article history: Received 23 February 2015 Received in revised form 8 May 2015 Accepted 11 May 2015 Available online 22 May 2015

The present study evaluates, at a proteomic level, changes in protein abundance in sunflower leaves in the absence or presence (at 50 or 700 mg) of cadmium (as CdCl2). At the end of the cultivation period (45 days), proteins are extracted from leaves with phenol, separated by two-dimensional difference gel electrophoresis (2-D DIGE), and excised from the gels. The differential protein abundances (for proteins differing by more than 1.8 fold, which corresponds to 90% variation) are characterized using nESI-LC–MS/ MS. The protein content decreases by approximately 41% in plants treated with 700 mg Cd compared with control plants. By comparing all groups of plants evaluated in this study (Control vs. Cd-lower, Control vs. Cd-higher and Cd-lower vs. Cd-higher), 39 proteins are found differential and 18 accurately identified; the control vs. Cd-higher treatment is that presenting the most differential proteins. From identified proteins, those involved in energy and disease/defense (including stress), are the ribulose bisphosphate carboxylase large chain, transketolase, and heat shock proteins are the most differential abundant proteins. Thus, at the present study, photosynthesis is the main process affected by Cd in sunflowers, although these plants are highly tolerant to Cd. & 2015 Elsevier Inc. All rights reserved.

Keywords: Sunflower Cadmium Proteomics 2-D DIGE LC–MS/MS

1. Introduction The contamination of soils and water with metals has created a major environmental problem, leading to considerable losses in plant productivity and hazardous health effects (Gratão et al., 2008). Exposure to toxic metals can intensify the production of reactive oxygen species (ROS), which are continuously produced in both unstressed and stressed plants cells. In this scenario, adapting to environmental changes is crucial for plant growth and survival, as the reaction products mentioned above can act in different ways in the cellular environment (Fridovich, 1995; Scandalios, 2005). The oxidative response in plants can be exacerbated by stressful conditions (Gratão et al., 2005). At the molecular level, the extent and nature of this response differs between species and n Corresponding author at: Spectrometry, Sample Preparation and Mechanization Group – GEPAM, Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-970 Campinas, SP, Brazil. Fax: þ55 19 3521 3023. E-mail address: [email protected] (M.A.Z. Arruda).

http://dx.doi.org/10.1016/j.ecoenv.2015.05.016 0147-6513/& 2015 Elsevier Inc. All rights reserved.

even between closely related varieties of the same species. Inside this context, cadmium is one of the most toxic metals, which affects plant growing and development, inducing the oxidative stress, resulting in a variety of antioxidant responses (Gratão et al., 2008). Besides this, genetic and cytotoxic effects were also noted in plants exposed to this metal, with inhibition of cellular division, alterations in the chromosomes, among others (Das et al., 1997). Additionally, oxidative stress also affects protein abundance, and comparative proteomics are currently used to evaluate stressed and non-stressed cultures at the protein level. As example, Garcia et al. (2009) evaluated the metal-ion stress in sunflower leaves, once that a mixed solution containing Cd, Cu, Zn and Pb was added to the culture. At this condition, a decrease of 50% on the quantity of proteins was observed, and this alteration was more pronounced for those proteins involved in energy metabolism (i.e. Rubisco, putative receptor protein kinase, oxygen-evolving enhancer protein 1, oxygen-evolving enhancer protein 2 and DEADbox ATP-dependent RNA helicase 3). Additionally, the authors present six new sequences of proteins to sunflower, which were

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inserted in the Expasy Proteomics Server, such as Putative receptor protein kinase, Cytochrome P450, Oxygen-evolving enhancer protein 1, DEAD-box ATP-dependent RNA helicase 3, Cell division protease ftsH homolog and Hypothetical protein 1. However, thus far, only two studies have evaluated the stress induced by adding metal to a culture from a proteomic point of view and using 2-D DIGE. The first study analyzed the effects of adding 20 mmol L  1 Cd to hydroponic cultures of poplar plants over 14 days (Kieffer et al., 2008). The protein content of the young poplar plant leaves was analyzed by 2-D DIGE, and proteins were identified using MALDI-TOF-TOF. Changes in abundance were detected for proteins involved in carbon metabolism, oxidative stress regulation, and pathogenesis. Because Cd had a low impact on the physiological parameters, the authors concluded that poplar trees could be used for phytoremediation. The second studied (Printz et al., 2013) combined phenotypical characterization and biochemical analyzes to evaluate the responses in the leaves and roots of young sunflowers to growth in hydroponic media contaminated with a mixture containing Cd (from 0.16 to 8.43 mg L  1), Ni (from 0.16 to 8.81 mg L  1) and Zn (from 1.82 to 98.09 mg L  1). The most affected proteins in the leaves were involved in the photosynthetic light reactions and carbon metabolism, whereas the most affected proteins in the roots were involved in respiration, maintaining the oxidative balance, protein and gene expression, and inducing programmed cell death. The authors of this study concluded that the use of sunflowers as a remediation tool for soils polluted with trace elements is limited. At the same time, our research group has recently demonstrated the high tolerance and resistance of sunflowers cultured in soil to Cd exposure (50, 350 or 700 mg per pot), indicating that sunflowers are hyperaccumulators; more than 900 mg kg  1 of Cd was found in the aerial compartments (steamþleaves) (Lopes Júnior et al., 2014). Then, put forward our studies related to Cd induced stress in sunflowers, the present manuscript increases our knowledge related to Cd-tolerance in sunflowers by evaluating the stress responses of cultured sunflowers to 50 and 700 mg Cd via comparative proteomics using 2-D DIGE and nESI-LC–MS/MS in tandem mode. Sunflowers could be useful for phytoremediation processes and could greatly benefit agribusiness, but little is known about the response of sunflower to stress regarding alterations in its proteome. Then, this work focuses on relevant information about differential proteins under sunflower stress.

2. Material and methods 2.1. Plant cultivation and cadmium exposure The IAC Iarama variety of sunflower (Helianthus annuus L.) from the Agronomical Institute of Campinas was used for this study (da Silva et al., 2011). Plants were cultivated in the greenhouse of the Department of Plant Biology at the University of Campinas, Brazil. Seeds were germinated in 770 mL plastic pots containing a 1:1 (w/w) mixture of sand and organic soil as a substrate. The plants were grown under three irrigation conditions: Control (only water), Cd-lower (50 mg of Cd) and Cd-higher (700 mg of Cd). Each group contained 12 plants. After seven days of growth (until the appearance of the first leaf), the plants were exposed to cadmium as a CdCl2 solution and cultivated for 45 days. The plants were grown under natural light at a temperature of 26 76 °C. Before flowering, the plants were harvested, divided into leaf, stem and root portions, and washed twice in distilled water. The leaves were immediately frozen in liquid N2 and stored in a  80 °C freezer (Forma 705, Thermo Scientific, Marietta, USA) to prevent protein unfolding and metal loss from their native structures.

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2.2. Comparative 2-D DIGE analysis 2.2.1. Protein extraction from sunflower leaves Proteins were extracted from the leaves of each plant group as previously described (Arruda et al., 2013; da Silva et al., 2010), with minor modifications. In brief, 1.0 g of sunflower leaves (fresh weight) was ground in a mortar in liquid nitrogen. Then, the plant material was homogenized and manually agitated for 10 min in 4.0 mL of extraction buffer containing 700 mmol L  1 sucrose, 50 mmol L  1 EDTA, 100 mmol L  1 KCl, 10 mmol L  1 thiourea, 50 mmol L  1 DTT, and 2 mmol L  1 PMSF dissolved in 500 mmol L  1 Tris–HCl at pH 7.5. The mixture was centrifuged (10 min, 6000g, 4 °C), and the supernatant was collected and subsequently extracted using the same protein extraction buffer saturated with phenol. The phenolic mixture was vortexed (10 min, room temperature) and then centrifuged (10 min, 6000g, 4 °C). After centrifugation, the phenol phase (upper phase) was collected and re-extracted twice in 5.0 mL of protein extraction buffer using the same protocol. The buffer phase was removed. The proteins contained in the phenol phase were precipitated overnight at  20 °C in 40 mL of 100 mmol L  1 ammonium acetate in methanol and then centrifuged (5 min, 8700g, 4 °C). The pellet was washed twice with 4 mL of 100 mmol L  1 cold ammonium acetate in methanol under centrifugation (8700 g, room temperature). The washed pellet was then dissolved in 300 mL of a rehydration buffer (7 mol L  1 urea, 2 mol L  1 thiourea, 4% (w/v) CHAPS and 20 mmol L  1 Tris at pH 8.8). After extraction, the protein concentration of the three samples was determined using the 2-D Quant protein assay kit (GE Healthcare, Uppsala, Sweden) before performing 2-D DIGE. 2.2.2. Protein labeling, electrophoretic separation and image analysis A comparative proteomics analysis of sunflowers leaves from the control and Cd-treated plants was performed using 2-D DIGE (Ettan DIGE, GE Healthcare, USA) as described previously (Arruda et al., 2013). 2-D DIGE gels from each group were prepared in triplicate, and two samples were evaluated by running. The protein precipitate was dissolved in lysis buffer (7 mol L  1 urea, 2 mol L  1 thiourea, 4% (w/v) CHAPS and 20 mmol L  1 Tris at pH 8.8) prior to dye labeling. Every step of the protein labeling was performed on ice and protected from light. First, each CyDye DIGE Fluor stock (GE Healthcare) was resuspended in 99.8% anhydrous N,N-dimethylformamide (DMF, Sigma, Germany). For each labeling reaction, 50 μg of the protein sample was incubated for 30 min with 400 pmol of a CyDye Fluor minimal dye, one of Cy3, Cy5 or Cy2 (Cy2 was used as an internal standard). To avoid preferential dye labeling, two biological replicates for each leaf sample were labeled with Cy3, and two were labeled with Cy5. In detail, 50 μg of each experimental sample was labeled with either Cy3 or Cy5. Equal aliquots (25 μg) of each sample were mixed to yield a pooled internal standard, and the samples were labeled with Cy2. The labeling reactions were stopped by adding 1 μL of 10 mmol L  1 lysine and incubation for 10 min on ice. After labeling, the samples were pooled and brought to 250 mL with rehydration buffer (7 mol L  1 urea, 2 mol L  1 thiourea, 4% (w/v) CHAPS, 0.002% (w/v) bromophenol blue, 0.5% (v/v) ampholytes and 1% (w/v) DTT). Samples were then applied to IPG strips (13 cm; pH 3–10; GE Healthcare) for passive rehydration overnight at room temperature. The rehydrated IPG strips were subjected to isoelectric focusing (Multiphor II system; GE Healthcare) at 14.6 kVh. After IEF, the strips were incubated in reducing equilibration buffer [50 mmol L  1 Tris–HCl pH 8.8, 30% (w/v) glycerol, 2% (w/v) SDS, 6 mol L  1 urea and 1% (w/v) dithiothreitol] for 15 min and subsequently in alkylation equilibration buffer (same as before, but 1% (w/v) dithiothreitol replaced by 2.5% (w/v) iodoacetamide) for 15 min. The IPG strips were then applied to 12.5%

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(w/v) sodium dodecyl sulfate (SDS) polyacrylamide gels that were cast between low-fluorescence glass plates and run on the EttanTM DALT system (GE Healthcare) at a constant voltage of 90 V and 25 mA per gel at 15 °C overnight in the dark. The differentially labeled, co-resolved proteome maps on each gel were imaged separately with dye-specific excitation and emission wavelengths using an Ettan DIGE Imager Scanner (GE Healthcare). The gel images were analyzed using the DeCyder 7.0 software (GE Healthcare). The DIA (differential in-gel analysis) module was used to define the spot positions, and the signals from the Cy3- and Cy5-labeled spots were normalized to the Cy2 signal of the internal standard. The DIA datasets for all of the gels from one sample were then collectively analyzed using the BVA (biological variation analysis) module, matches protein spot patterns across gels, followed by statistical analysis of the matched spots. A regulation factor of 1.8 (90% variation) was used to determine differential variations in protein abundance. Statistical significance was assessed using Student's t-test based on the log abundances of the standardized proteins. After image analysis, gels from the leaves of each plant group were prepared by loading Z300 μg of protein using the same separation conditions previously described for 2-D DIGE. These gels were fixed in 10% (v/v) acetic acid and 40% (v/v) ethanol, stained with Colloidal Coomassie Blue G250, and scanned with an ImageScanner II (Amersham Biosciences, Uppsala, Sweden) with the densitometer operating at 300 dpi resolution. Images were analyzed using the Image-Master 2D Platinum 6.0 software (GeneBio, Geneva, Switzerland) (da Silva et al., 2010). 2-DE and 2-D DIGE maps were matched to locate the spots of interest assigned by the DIGE analysis. These spots were excised manually and subjected to MS for protein identification. 2.3. Protein identification by LC–MS/MS 2.3.1. In-gel protein digestion The protein spots of interest identified by the DIGE study were excised manually from the Coomassie-stained gels and subjected to in-gel trypsin digestion, as described previously (Arruda et al., 2013; da Silva et al., 2010). Briefly, the spots excised from the gels were placed into a micro-SPE plate containing peptide affinity resin, and the digestion and vacuum elution protocols were performed according to the manufacturer's recommendations using the Montages In-Gel digestZP kit (Millipore, Bedford, USA). First, each protein spot was destained with acetonitrile, and trypsin was subsequently added. For in-gel trypsin digestion, each spot was incubated with 166 ng of trypsin for 3 h at 37 °C. Then, the peptides were extracted from the resin twice using 0.1% (v/v) TFA in 50% (v/v) ACN. The vacuum elution was performed using a Multiscreens Vacuum Manifold (Millipore) system. 2.3.2. Mass spectrometry analysis After trypsin digestion, the peptides obtained were dried and resuspended in deionized water. Then, the resulting peptide mixture was analyzed by LC–MS/MS using a nanoAcquity UPLC (Waters Co., Manchester, UK) coupled to a Waters Synapt HDMS (Waters Co.) mass spectrometer equipped with a BEH C18 column (100 mm  100 mm, Acquity Waters) and operated on a gradient from 2–90% acetonitrile in 0.1% (v/v) formic acid with flow rate of 1.0 μL min  1 for 40 min. The mass spectrometer was handled by the Data Dependent Analysis (DDA) software, which acquires one spectrum per second. When multi-charged species were detected, the three most intense species were fragmented in the collision cell (collision energy set according to precursor m/z and charge). The spectra acquisition was performed using the MassLynx v.4.1 software. All files from the acquired mass spectra were converted to peak list format using Mascot Distiller (Matrix Science) and searched

against the National Center for Biotechnological Information (NCBI) and Swiss-Prot protein databases. The MASCOT Server 2.3 MS/MS search engine was used with the following parameters for protein identification: carbamidomethylation of cysteine as the fixed modification, oxidation of methionine as the variable modification, one missed trypsin cleavage, and a tolerance of 70.1 Da for the precursor and fragment ions. The significance threshold was set at p o0.05, which corresponds to a minimum score of 30.

3. Results and discussion 3.1. Comparative proteomics of Helianthus annuus L. by 2-D DIGE Cadmium is not involved in any essential biological functions in living organisms. In plants, cadmium is associated with various metabolic disorders such as nutritional imbalance, photosynthetic apparatus damage, and oxidative stress (Azevedo et al., 2012; Sytar et al., 2012). The sunflower is recognized for its cadmium resistance, tolerance and hyperaccumulation (Cutright et al., 2010; Garcia et al., 2009; Lopes Júnior et al., 2014). Cadmium toxicity effects were observed in sunflower plants at both the macroscopic and the molecular level. At the macroscopic level, chlorotic areas were observed in leaves during growth, and necrotic areas appeared in the basal part of the stems, when plants were treated with 700 mg of Cd. However, sunflowers that were exposed to 50 mg of Cd presented no pronounced symptoms of Cd toxicity (data not shown). At the molecular level, the presence of high levels of Cd in soil significantly affects the protein content of the leaves. Control plants displayed 518 73 mg g  1 of protein, whereas plants receiving the Cd at lower and higher concentrations presented 596 727 mg g  1 and 308 716 mg g  1 of protein, respectively. Two factors possibly contribute to this effect: (1) a decrease in protein synthesis, and (2) an increase in leaf proteolytic activity due to Cd-induced oxidative stress, promoting the accumulation of oxidatively damaged proteins. For example, the proteolytic activity of pea plant leaves (Pisum sativum L.) was found to increase by 20% upon Cd treatment (Romero-Puertas et al., 2006). To evaluate Cd toxicity at the molecular level, the proteomes of sunflower leaves exposed to different treatments were assessed using 2-D DIGE. The protein spot map of each sample is visualized in this technique, and changes in individual protein abundance are detected and quantified. In 2-D DIGE analysis, two samples are labeled with spectrally resolvable fluorescent dyes, mixed prior to IEF, and resolved on the same 2-D gel (Arruda et al., 2011). Therefore, this technique eliminates the experimental variation observed in conventional 2-D electrophoresis and provides greater confidence to comparative proteomic studies (Arruda et al., 2011; Rabilloud et al., 2010). In a previous study carried out by our research group (da Silva et al., 2010), protein extracts from sunflower leaves were separated by 2-D PAGE, and protein spots were observed over a wide pI range. Thus, a pH range from 3 to 10 was adopted. By comparing the proteomic profiles of sunflower leaves from plants exposed to different treatments (Fig. 1), we observed that cadmium toxicity also affects the primary metabolism of plants. The toxic effects were more pronounced in plants treated with 700 mg Cd, resulting in a large number of proteins with differential abundance (Fig. 1B). The criteria used to determine whether proteins were differentially abundant were a regulation factor of 1.8 (or 90% variation) and p o0.05 following Student's t-test. Five proteins (all of low abundance) were found to be differentially abundant between the Control vs. Cd-lower groups, whereas 19 proteins were detected with abundance changes between the Control vs. Cd-higher groups (4 with higher abundance and 15

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with lower abundance). The influence of cadmium levels on the leaf proteome of Cdtreated plants was also investigated by comparing protein abundance profiles. As observed in Fig. 2, 15 proteins showed a significant change in their abundance (3 higher and 12 lower) in a comparative study between the Cd-lower vs. Cd-higher plant treatments. These results were similar to those obtained when the proteomic profiles of sunflower leaves from the Control and Cdhigher groups (Fig. 1B) were compared. In the present study, cadmium significantly affected the primary metabolism of sunflowers, but the intensity of the toxic effects depended on the level of Cd exposure. Thus, plants treated with 700 mg of Cd displayed lower protein content and a greater number of proteins with changes in abundance compared with plants in the other treatment groups. Worth mentioning the changes in the proteome of plants caused due to the presence of chlorine ions are minimal, once that the level of chlorine added to sunflowers of the Cd-higher group was 0.15% only. The toxic effects of chlorine ions in sunflower metabolism, such as inhibition of germination, growth of reduction and oxidative stress are observed at an exposure level exceeding 0.8% (Jabeen and Ahmad, 2013). 3.2. Identification of differentially abundant proteins

Fig. 1. Representative two-dimensional difference gel electrophoresis (2-D DIGE) maps of the proteome of sunflower leaves. The gels show (A) Control vs. Cd-lower and (B) Control vs. Cd-higher. Delimited spots correspond to proteins with changes in abundance: red indicates decreased abundance, and blue indicates increased abundance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Representative two-dimensional difference gel electrophoresis (2-D DIGE) maps of the proteome of sunflower leaves. The gel shows Cd-lower vs. Cd-higher. Delimited spots correspond to proteins with changes in abundance: red indicates decreased abundance, and blue indicates increased abundance. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To identify the protein species corresponding to the differentially abundant spots detected and quantified by 2-D DIGE, the proteomic study was followed by nESI-LC–MS/MS. Preparative gels for each leaf sample were stained with Colloidal Coomassie Blue G250 and matched to the 2-D DIGE data. All spots of interest were located, manually excised, subjected to in-gel trypsin digestion and analyzed. The identified proteins are presented in Table 1. The observed experimental pI/MW values are similar, indicating that the protocol used produced accurate results. To increase our understanding of the roles of proteins involved in the Cd stress response, the proteins identified were categorized into six major groups based on their biological function according to information provided by NCBI, UniProt and Bevan et al. (1998). The functional classification of the identified proteins can be observed in Table 1. As expected for a study of plant leaves, the majority of the proteins were classified into the energy category, and nearly half of the proteins were distributed between the disease/defense, protein destination and storage, signal transduction, and transporter categories. Additionally, the interactions between some identified proteins were evaluated using the STRING 9.1. The photosynthetic machinery of higher plants is very sensitive to Cd presence. In the present study, most proteins involved in photosynthesis showed decreased abundance in Cd-treated sunflowers. Rubisco is an enzyme that catalyzes the CO2 assimilation reaction in plants (the first step of Calvin cycle); multiple spots containing the large and small subunits were identified, and Rubisco proteolytic fragments were found less abundant when considering Cd-treated plants. These results suggest that Cd stress can promote the inactivation and degradation of this enzyme. Polatajko et al. (2011) demonstrated that cadmium impairs the structure of Rubisco by displacing the magnesium cofactor. In addition, Hajduch et al. (2001) reported an increase in the number of Rubisco proteolytic fragments in metal-stressed rice leaves that was associated with a higher formation rate of reactive oxygen species (ROS). Regarding the work developed by Printz et al. (2013), Rubisco LSUs and SSUs were also found differentially abundant in sunflower leaves, as response of a polymetallic (Cd þ Znþ Ni) treatment. In addition to Rubisco, transketolase is involved in the Calvin cycle and catalyzes the reaction that regenerates the CO2 acceptor molecule ribulose-1,5-bisphosphate (Flechner et al., 1996). Our study found a decrease on the abundance of

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Table 1 Proteins in sunflower leaves with changes in abundances ( 41.8 fold – 90% variation) after Cd stress. Spot no.

2 10 12 5 6 7 8 9 2 3 17 13

9 10 15

1 6

Control vs. Cd-lower Functional category: energy Ribulose bisphosphate carboxylase small chain, chloroplastic [Helianthus annuus] Functional category: signal transduction Nucleoside diphosphate kinase B [Flaveria bidentis] Control vs. Cd-higher Functional category: energy Ribulose bisphosphate carboxylase large chain [Helianthus annuus] Ribulose bisphosphate carboxylase large chain [Helianthus annuus] Transketolase, chloroplastic [Spinacia oleracea] Transketolase, chloroplastic [Spinacia oleracea] Transketolase, chloroplastic [Solanum tuberosum] Transketolase, chloroplastic [Solanum tuberosum] Transketolase, chloroplastic [Spinacia oleracea] Functional category: disease/defense Stromal 70 kDa heat shock-related protein, chloroplastic (fragment) [Spinacia oleracea] Stromal 70 kDa heat shock-related protein, chloroplastic [Pisum sativum] 17.6 kDa class I heat shock protein [Helianthus annuus] Functional category: transporters ATP synthase subunit alpha, chloroplastic [Helianthus annuus] Cd-lower vs. Cd-higher Functional category: energy Ribulose bisphosphate carboxylase large chain [Helianthus annuus] Ribulose bisphosphate carboxylase large chain [Helianthus annuus] Ribulose bisphosphate carboxylase small chain, chloroplastic [Helianthus annuus] Functional category: protein destination and storage Glycinin G4 [Glycine max] Functional category: signal transduction Chain A, Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase [Arabidopsis thaliana]

Protein accession number

Score

Matched peptide

% sequence coverage

Theoretical pI/MW (kDa)

Experimental pI/MW (kDa)

Volume average ratio

t-test value

RBS_HELAN

70

5

30

9.07/20.5

9.45/16.6

 1.85

0.29

NDKB_FLABI

167

10

47

6.43/16.2

7.26/16.3

 2.07

0.13

RBL_HELAN

130

13

26

5.95/54.3

5.83/68.1

 1.92

0.016

RBL_HELAN

320

20

33

5.95/54.3

5.76/56.5

 1.80

0.0034

TKTC_SPIOL TKTC_SPIOL TKTC_SOLTU TKTC_SOLTU TKTC_SPIOL

137 118 123 150 126

6 5 8 7 6

10 7 10 10 10

6.20/80.7 6.20/80.7 5.94/80.3 5.94/80.3 6.20/80.7

5.56/77.4 5.64/75.8 5.74/77.4 5.81/78.6 5.87/80.2

 1.95  1.98  1.93  2.06  3.30

0.000094 0.000072 0.00053 0.00036 0.0015

HSP7S_SPIOL

35

5

8

4.87/64.9

4.80/69.5

 2.10

0.0043

HSP7S_PEA

65

8

13

5.22/75.6

4.92/72.0

 1.83

0.0021

HSP11_HELAN

39

4

15

5.25/17.6

4.67/16.6

1.98

0.0015

ATPA_HELAN

36

3

4

5.20/55.5

4.82/63.4

 1.90

0.021

RBL_HELAN

141

17

30

5.95/53.8

6.02/56.2

 1.91

0.012

RBL_HELAN

337

19

25

5.95/53.8

5.85/55.1

 1.96

0.0084

RBS_HELAN

138

7

43

9.07/20.5

9.19/15.6

2.14

0.0012

GLYG4_SOYBN

30

3

5

5.29/64.0

6.03/66.0

 3.01

0.0019

gi|380259052

51

4

3

6.13/55.5

6.41/76.2

 1.91

0.0070

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4

Identified protein (species)

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transketolase under Cd cultivation, and, together the Rubisco decrease in abundance, the reduction on CO2 fixation efficiency under Cd stress is then suggested. Significant changes in the abundance of nucleoside diphosphate kinase (NDPK) were found under Cd stress in sunflowers. This protein is involved in cell signaling and energy metabolism and it is responsible for maintaining the nucleotide balance in cells. In previous studies (Roberts et al., 1997), NDPK was reported to cooperate metabolically in the mitochondria, while Johansson et al. (2008) demonstrated that an interaction between the proteins modulates enzyme activity – adenylate kinase stimulated NDPK activity. In our study, the NDPK was less abundant in plant leaves treated with 50 mg Cd, which is most likely as general stress response mechanism. Another enzyme involved in intracellular signaling and phosphate group transfer, Inositol-pentakisphosphate 2-kinase, also showed a decrease in abundance during stress in the Cd-higher plants group. These events showed demonstrate that Cd stress promotes imbalances in the cells' energy status in Cd-treated sunflowers. Few protein spots were showed significant changed in abundance corresponding to proteins species associated with defense against Cd-induced stress. Plants under normal environmental conditions produce ROS; the major ROS in plants are the superoxide radical (O2  ), hydrogen peroxide (H2O2), singlet oxygen (1O2) and the hydroxyl radical (HO2  ) (Rodziewicz et al., 2013). These ROS play an important role in signaling and are involved in the activation of defense responses. However, these species can also oxidize various molecules and cellular structures (Johansson et al., 2008). To minimize potential damage, plants have developed various antioxidant systems to balance the production and concentration of ROS. The presence of cadmium in the organism induces imbalances in the antioxidant system, leading to high ROS production (Gratão et al., 2005). Important proteins involved in stress responses identified in our study include some heat shock proteins (HSPs or chaperones), such as stromal 70 kDa heat shockrelated protein and 17.6 kDa class I HSP. In plants, the HSPs are responsible for maintaining cellular homeostasis both under optimal growth conditions and under stress by correcting the folding, translocation and degradation of proteins (Gupta et al., 2010). These proteins are classified based on their molecular weight and function; HSP70 is involved in the transport of proteins into and within chloroplasts, enabling the biogenesis and function of this organelle (Johansson et al., 2008). In addition, small HSPs (sHSPs; 15–40 kDa) are quite diverse and abundant in plants, but their exact mechanisms of action remain unknown. However, their production is associated with protection of subcellular structures in plant cells against different types of stress (Rodziewicz et al., 2013). The chloroplast sHSP protects photosystem II and the thylakoid membranes of Chenopodium album under Cd stress (Haq et al., 2012). In the present study, only HSP17.6 showed an increase in abundance in the leaves of Cd-treated plants. Kieffer et al. (2008) observed that a group of HSPs, particularly HSP70, was significantly decreased in poplar leaves following exposure to cadmium stress. This decrease in the abundance of several important enzymes related to ROS scavenging could contribute to the general imbalance of ROS generation/detoxification that leads to oxidative stress. Cd toxicity also affected proteins involved in the transport of ions and molecules between cell organelles in leaves; all of the identified proteins showed a lower abundance in sunflower leaves under Cd stress conditions. The alpha subunit of ATP synthase regulates H þ transport in the enzyme complex ATP synthase. This complex, which is located in the chloroplasts, is composed of multiple subunits such as beta, gamma, delta, epsilon, a, b, b′ and c, and synthesizes ATP from ADP and Pi (Yoshida et al., 2001). The Calvin cycle uses ATP and NADPH to synthesize ribulose

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biphosphate, which reacts with CO2 in a reaction catalyzed by Rubisco. Tezara et al. (1999) demonstrated that decreases in CO2 assimilation in sunflower leaves under water stress correlated with disturbances in the ATP synthase complex and not in the activity of ribulose bisphosphate carboxylase/oxygenase (Rubisco). Therefore, the inhibition of ribulose bisphosphate synthesis is the limiting factor in CO2 assimilation. In addition to H þ transport, sucrose transport can be impaired in Cd-treated plants because the abundance of sucrose-binding protein is significantly reduced in Cd-higher group. In addition to their roles in plant seeds, glycinins can be found in other compartments such as the roots, stems and leaves (Utsumi et al., 1993). These storage proteins are composed of six subunits that each contain an acidic polypeptide chain (36– 40 kDa) linked via disulfide bonds to a basic polypeptide chain (18–20 kDa) (Riblett et al., 2001). The abundance of all of the glycinins was significantly reduced in the leaves of Cd-treated plants, and glycinin G4 was less abundant in plants treated with 700 mg Cd. Transcription factors are proteins that regulate gene control. TF factors have the ability to control the expression of target genes by binding to specific sequences in these genes (Nakashima et al., 2009). The abundance of several proteins involved in transcription has been reported to change in response to salt and drought stress (Rodziewicz et al., 2013). In our study, the abundance of inositol 1,3,4,5,6 – pentakisphophate 2-Kinase decreased under Cd stress. 3.3. Interactions between some proteins identified When considering protein–protein interactions (Fig. 3), and using the Arabidopsis thaliana as a model organism, once that there is no information inside the stringprotein program for H. annuus, this program showed the results of five proteins (i.e. Rubisco, ATP binding – heat shock proteins, inositol kinase, transketolase and ATP synthase alpha chain) from those differentially found and identified proteins in our study (see Table 1). From these proteins, three different clusters are observed (see Fig. 3), indicating a close correlation between, at least, three proteins identified. These proteins are heat shock proteins, ATP synthase alpha chain and Rubisco. It is interesting, once that these proteins are involved in the ATP synthesis and transport of protons. The ATP synthesis of utmost important once that provides the necessary energy to a cell through the ADP þinorganic phosphorus, both being joined together by the ATP synthase. As all of these proteins are at lower abundance (see Table 1) in our study, a lower production of Rubisco was also observed (see also Table 1), corroborating our results. Rubisco is involved in the ATP binding, and at the end, in the energy metabolism of plants. As observed during the cultivation, the development of plants belonging to Cd-higher treatment was prone to be worst, as already discussed in our previous work (Lopes Júnior et al., 2014). Another cluster is that one where the transketolase is linked. This protein is involved in the response to Cd and located in the chloroplast stroma. This fact may also be linked with the identification of the stromal 70 kDa heat shock-related protein (see Table 1). In the network of Fig. 3, the transketolase is extremely linked to some transaldolases, which are also involved in the response to Cd. In fact, the link between the transketolase and transaldolases also suggests that due to the excess of Cd in the Cd-higher treatment, the catabolic metabolism for lactate and acetate was modified. The last, and the smallest, cluster is formed by the nucleoside diphosphate kinase and the ATP binding thymidylate kinase, which encodes thymidylate kinase. Again, the energy metabolism is involved in such cluster. According the analysis of the identified proteins, through the stringprotein program, some of the most important biological

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Fig. 3. Protein interaction network generated and visualized with STRING 9.1 for protein identified in our study. The codes represent: CR88 – Heat shock protein; AT2G07698 – ATP synthase; AT2G33040 – ATP synthase gamma chain; ATPQ – ATP synthase d chain; ATPC2 – enzyme regulator; ATPC1 – enzyme regulator; IPK2a – Inositol kinase; AT1G73110 – Rubisco; EMB2107 – Embryo defective 2107; AT5G2000–26S proteasome AA-ATPase subunit; RPN5B – Regulatory particle non-ATPase subunit 5B; ZEU1 – ATP binding/thymidylate kinase; AT1G17410 – Nucleoside kinase; AT5G13420 – Transaldolase; AT2G45290 – Transketolase; AT1G12230 – Transaldolase.

processes are summarized, as follow: in the cellular process, four differential and identified proteins are involved, such as heat shock protein, inositol kinase, and Rubisco. To the primary metabolic process, cellular component organization, organic substance metabolic process and metabolic process, heat shock protein, inositol kinase and Rubisco are involved, and finally, in the response to stimulus are involved both transketolase and heat shock protein.

Acknowledgments The authors thank the Fundação de Amparo a Pesquisa do Estado de São Paulo (2012/21344-7), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, Brazil, 303188/2011-1) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília, Brazil, 189085) for financial support and fellowships.

4. Conclusion Appendix A. Supplementary information A proteomic study using 2-D DIGE and mass spectrometry was performed to increase our understanding regarding biochemical bases of the Cd toxicity mechanisms in sunflower metabolism. A sample preparation procedure based on phenol extraction, combined with the electrophoretic conditions, allowed sufficient gel resolution to visualize a large number of protein spots from sunflower leaves. Additionally, the biological processes that were most affected by Cd could be identified by evaluating the proteins that were most affected. Most differential abundant proteins were found when considering control vs. Cd-higher treatment, with 11 proteins identified. In fact, proteins involved in energy and disease/defense (including stress), such as ribulose bisphosphate carboxylase large chain, transketolase, and heat shock proteins, were the most affected by Cd exposure, as determined by a regulation factor of 1.8. By analyzing the interaction between the proteins identified as well as others presented in the protein network, some process were found to be differential, such as primary metabolic process, cellular component organization, organic substance metabolic process and metabolic process, cellular process and response to stimulus. As conclusion, photosynthesis is the main process affected by the presence of high Cd concentrations in sunflowers, although the plants are highly tolerant to Cd exposure, as only minor changes were observed in their physiological processes. Therefore, proteomic results from this study are in agreement with those from our ionomic approaches (Lopes Júnior et al., 2014).

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ecoenv.2015.05. 016.

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