Identification of Agrostis tenuis leaf proteins in response to As(V) and As(III) induced stress using a proteomics approach

Plant Science 176 (2009) 206–213

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Identification of Agrostis tenuis leaf proteins in response to As(V) and As(III) induced stress using a proteomics approach Isabelle Duquesnoy a,b, Pascale Goupil a, Isabelle Nadaud c, Ge´rard Branlard c, Agne`s Piquet-Pissaloux b,*, Ge´rard Ledoigt a a b c

ERTAC-PIAF, UMR INRA-UBP, Tumeurs et Autosurveillance Cellulaire, 24 avenue des Landais, 63177 Aubie`re Ce´dex, France De´partement Agricultures et Espaces, ENITA C, 63370 LEMPDES, France UMR, INRA GDEC-UBP, 63100 Clermont-Ferrand cedex, France

A R T I C L E I N F O

A B S T R A C T

Article history: Received 12 July 2008 Received in revised form 5 October 2008 Accepted 6 October 2008 Available online 25 October 2008

Agrostis tenuis is known to be able to metabolise arsenate (As(V)) and arsenite (As(III)) which are toxic salts for most plants. A proteomic approach was developed to identify proteins expressed in response to treatments with these salts. A. tenuis plants were grown hydroponically in the presence of 134 and 668 mM As(V) or As(III) for 8 days at pH 7. During arsenic treatments, leaves showed chlorotic symptoms but fresh and dry leaf weights were not reduced, except in the presence of 668 mM As(III). On the contrary, a slight increase in biomass was observed with high As(V) concentrations. Thus, A. tenuis was more sensitive to As(III) than to As(V) and biomass was affected. Proteomic analysis enabled identification of a set of A. tenuis leaf proteins differentially expressed in response to arsenic exposure including a major functionally homogeneous group of enzymes such as oxygen-evolving enhancer protein, RuBisCO small and large subunits, RuBisCO activase and ATP synthase involved in the Calvin or Krebs cycle. The adaptative response to treatments resulted in partial disruption of the photosynthetic processes with prominent fragmentation of the RubisCO. Other proteins expressed differently from controls were identified and are possibly involved in the tolerance mechanisms of A. tenuis to arsenic treatments. ß 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Agrostis tenuis Arsenate Arsenite Leaf proteome Ribulose-1,5-bisphosphate carboxylase/ oxygenase

1. Introduction Soil contamination with toxic heavy metals and metalloids has become a worldwide concern. Arsenic contamination of ground and surface waters and soils has frequently been reported in many places around the world [1,2]. Inorganic arsenate [As(V)] and arsenite [As(III)] are the main soluble arsenic species found in both soil and water; whereas As(V) predominates under aerobic conditions, As(III) is the dominant form under anaerobic conditions [1]. Arsenic contamination is associated with increased risk of certain types of human cancer such as skin, bladder, lung, kidney and liver cancers [3]. Thus, the maintenance of safe levels of water and soil arsenic is a very important environmental task. As(III), which is generally considered as the most phytotoxic arsenic species, seems to act by binding to sulfhydryl groups of

* Corresponding author. E-mail address: [email protected] (A. Piquet-Pissaloux). Abbreviations: BRGM, Bureau de Recherche Ge´ologique et Minie`re; CHAPS, 3-3cholamidopropyl dimethyl-ammonio-1-propane sulfonate; MALDI-TOF, matrix assisted laser desorption ionization-time of flight. 0168-9452/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2008.10.008

enzymes and proteins, thus leading to the disruption of cellular function and death, while As(V) interferes with phosphate uptake, metabolism, and phosphorylation reactions by acting as a phosphate analog [4–6]. A few plants have been reported to be able to hyperaccumulate arsenic, including some species from the genera Pteris, Agrostis, Paspalum, Jasione and Lemma [7,8]. These natural attenuation species accumulate considerable levels of arsenic [9] which means they are of more interest in phytoextraction protocols than sensitive plants. However, they display poor growth and biomass production rates, thus rendering their use in phytoremediation strategies more difficult. Agrostis tenuis is a monocotyledon that is recognized to be a tolerant species that behaves as a pseudometallophyte accumulator [10] and has been shown to be able to grow in smelter waste in England and accumulate arsenic up to 1% of its dry weight [11]. Arsenic tolerance in hyperaccumulator plants has been the subject of recent studies [12,13] but the tolerance reactions towards arsenic exposure remain poorly understood in higher plants. Plant responses to heavy metal toxicity may take different forms: biochemical tolerance (modifications in photosynthesis, Reactive Oxygen Species (ROS) production) [14], detoxication

I. Duquesnoy et al. / Plant Science 176 (2009) 206–213

mechanisms (phytochelatins or metallothioneins and cellular compartmentation) [15] and competition for nutrient compounds such as phosphorus [16,17]. Techniques such as transcriptomics-based DNA-microarrays [18] and proteomics have recently been used to evaluate response to environmental factors. The proteomics approach is a powerful tool for describing the functional status of different cellular compartments or organs [19]. This technique including twodimensional electrophoresis (2-DE) of extracted proteins, image analysis, mass spectrometry and data base interrogation can be used for analysing proteomes affected by different environmental conditions, such as those resulting from the exposure to arsenic [20,21]. The aim of the present study was to identify both the physiological and biochemical processes that occur in A. tenuis plant following exposure to either As(V) or As(III) for 8 days at two concentrations (134 and 668 mM) in physiological pH conditions. The arsenic concentrations were selected according to those found in polluted sites in the Auvergne region, France [22] and in works by Tu and Ma [16]. First, the effects of As(V) and As(III) on biomass production in A. tenuis were measured. Proteins extracted from leaves were analyzed using 2-DE and the resulting patterns were compared using image analysis software. Differentially expressed spots were then digested and peptide mass fingerprints obtained with matrixassisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) were used for protein identification. 2. Materials and methods 2.1. Plant growth and treatments Seeds of A. tenuis were grown in vermiculite in a greenhouse with a light/dark photoperiod of 16/8 h at 25 8C. After 1 month of growth, 240 healthy plants were transferred in a Hoagland nutrient solution made with deionized water, containing the different As(V) or As(III) concentrations, in the light/dark photoperiod of 16:8 h, at 25 8C and at pH 7. After 8 days of hydroponic acclimation, A. tenuis leaves were harvested for triplicate determination of fresh and dry weights, and for proteomic studies. Fresh leaf weight was measured immediately after harvesting and the leaves were placed in an oven for 4 days at 50 8C for determination of dry weight. 2.2. Protein extraction After arsenic treatments, total soluble proteins were extracted from A. tenuis leaves with three independent replicates for each treatment, as previously described [23–25]. The fresh samples (300 mg) were ground in liquid nitrogen and homogenized in 1 ml of a glacial acetone solution containing 0.07% (v/v) 2-mercaptoethanol, 0.34% (w/v) plant protease inhibitor cocktail (Sigma Steinhein, Germany) and 4% (w/v) polyvinyl pyrolidone (Sigma– Aldrich, France) for 2 h at 4 8C. After centrifugation at 15 000  g, for 15 min at 4 8C, the pellets were washed with ice-cold acetone solution and centrifuged again at 4 8C. This step was repeated three times and the pellets were then dried at room temperature and stored at 80 8C for later use. The sample powder was then solubilized in lysis buffer [4% (w/v) CHAPS, 7 M urea, 2 M thiourea, 2% (w/v) dithiothreitol (DTT), 1% (w/v) Pharmalytes pH 3–10 and 1% (w/v) Resolytes pH 6–9.5] for 90 min at room temperature. This suspension was sonicated at 20 W for 2 min, followed by a final incubation for 30 min at room temperature. Protein extracts were centrifuged at 10 000  g for 20 min at 4 8C and the protein concentration was determined on supernatants by the Bradford

207

assay (Bio-Rad) with Bovine Serum Albumin (BSA) as standard [26]. 2.3. Protein electrophoresis and staining Isoelectric focusing (IEF) of 150 mg total protein was carried out on an immobilized pH gradient (IPG) 18 cm pH 3–10 linear strips in a Pharmacia Multiphor II cell (Amersham Pharmacia Biotech, Uppsala, USA). The running condition was performed at 15 8C as follows: 0.3 kV for 1 h, 2.9 kV for 1 h 30 min, and 56.8 kV for 20 h giving a total of 60 kVh. The focused strips were equilibrated twice in solution. The first balance was performed for 10 min in 50 ml solution containing 6 M urea, 50 mM Tris–HCl (pH 8.8), 30% (v/v) glycerol, sodium dodecyl sulfate (SDS) and 2% (w/v) DTT. The second steady-state was obtained for 15 min in the solution modified by the replacement of DTT by 4 vinyl pyridine for alkylation. Separation in the second dimension was performed by SDS Polyacrylamide Gel Electrophoresis (SDS PAGE) in a vertical slab of acrylamide (14% total monomer, with 2.1% crosslinker) using a Hoefer apparatus (Amersham). The analytical 2D gels were stained with Coomassie Brilliant Blue as described by Neuhoff et al. [27] with the modified cations proposed by Rabilloud and Chevallet [28]. 2.4. Image analysis Six analytical replicates for each treatment were analyzed and scanned. Spots were quantified using Melanie-5 software (GE Health care software). After image treatment, spot detection, protein quantification (%spot volume) and spot pairing were carried out based on Melanie-5 default settings. Then, spot pairs were investigated visually and the scatter plots between gels of each data point were displayed to estimate gel similarity and experimental errors. The molecular mass (MM) and the isoelectric point (pI) of proteins were determined by co-electrophoresis of standard protein markers (Amersham Pharmacia Biotech). The spots, whose volume varied significantly between controls and arsenic treated plant extracts, were excised for trypsin digestion. 2.5. Statistical analysis Student’s test was used to compare the means of percent volume of spots for determination of significant differences between treated samples versus controls. The data were written as the mean  SD. Spots were determined to be significantly up- or down-regulated when P  0.05. 2.6. Trypsic digestion Gel spots were excised, incubated for 30 min in 25 mM NH4HCO3, 5% (w/v) acetonitrile (ACN) then washed twice in 25 mM NH4HCO3—50% (w/v) ACN for 15 min and then dehydrated with pure ACN for 10 min before drying in a Speed Vac Concentrator (Bachofer). Trypsic digestion was performed overnight at 37 8C, with 150 ng of Trypsin (Promega, Madison, WI, USA) in 25 mM NH4HCO3. Peptides were extracted by adding 33% (v/v) ACN containing 0.1% trifluoroacetic acid. The trypsin peptide mixture then was ready to use for MALDI-TOF analysis. 2.7. Identification of A. tenuis leaf proteins by MALDI-TOF mass spectrometry One microliter of ‘‘ready to use’’ trypsin peptide mixture was loaded onto the target and crystallised with an equal volume of acyano-4-hydrocinnamic acid matrix solution (5 mg/ml in 50%

I. Duquesnoy et al. / Plant Science 176 (2009) 206–213

208

ACN/0.1% (v/v) trifluoroacetic acid). After drying at room temperature the peptide mixture was analyzed using a MALDITOF mass spectrometry Voyager DE super STR (Applied Biosystems, Fragmingham, MA, USA). External calibration was performed using standard mass peptide solutions (Proteomix, LaserBio Labs, SophiaAntipolis, France). Spectra were also internally calibrated using trypsin autolysis products. The peptide mass fingerprint obtained from each sample was analyzed and the database analysis was performed using MASCOT software (Matrix science, London, UK, http://www.matrixscience.com). When Mascot failed to provide identification, spectra were interpreted using sequencing algorithms, with the derived amino acid sequences subjected to NCBI and SWISSPROT databases. The search criteria used were one missing trypsin cleavage site, partial methionine oxidation, partial pyrediethylation of cysteine, and a mass deviation lower than 50 ppm. Preliminary experiments indicated that the direct extraction of proteins with lysis buffer produced few proteins and poor resolution on 2-DE IPG  SDS PAGE (data not shown) due to interfering compounds such as polyphenols, terpenes and other organic acids in leaves. Hence, a protocol including precipitation with polyvinyl pyrolidone and sonication was optimized to obtain adequate resolution of A. tenuis leaf proteins. Protein extracts were first fractionated in a pH range of 3–10 followed by SDS PAGE with 14% polyacrylamide concentration (Fig. 2). Most leaf proteins were located within a narrow pH range (5–7) and only a few proteins were detected at extreme pH regions of 3–4 or 9–10. The optimized procedure increased protein resolution since polypeptides were separated into an appropriate range between 14 and 43 kDa. The distribution patterns of spots presented highly abundant proteins migrating between 14–23 and 30–43 kDa with multiple pI values. 3. Results

Fig. 1. Biomass changes in metal stressed A. tenuis leaves. (A) Fresh leaves and (B) dried leaves. Table 1 Average number of protein spots revealed after 2D IPG–SDS PAGE of leaves submitted to arsenic treatments. Total numbers of up- or down-regulated spots were obtained after matching between control and arsenic gels. Results are means of three independent replicates.

3.1. Physiological response to arsenic After 8 days of arsenic treatments, A. tenuis supplemented with 134 mM As(V) or As(III) showed chlorotic symptoms while control plants remained healthy (data not shown). Plants treated with 668 mM As(V) or As(III) developed a phenotypical response with yellow leaves and necrotic lesions that were higher with As(III). The presence of 134 mM As(V) or As(III) in hydroponic cultures led to a slight increase in leaf fresh biomass compared to control plants, while leaf dry weights showed no significant change (Fig. 1). However, the highest concentration of arsenic (668 mM) triggered changes in plant biomass for both arsenic species. The As(V) treatment increased leaf fresh weight while As(III) treatment clearly reduced it. Similar results were observed for leaf dry weight. These results showed that A. tenuis plants were more sensitive to As(III) and that biomass was affected. 3.2. Two-dimensional gel electrophoresis analysis Melanie-5 analysis of three replicated gels showed that 381 proteins were reproducibly detected in control Coomassie-stained

Total number of spots Spots disappearance Up-regulated (score > 60) Up-regulated (score < 60) Down-regulated (score > 60) Down-regulated (score < 60)

Control

As(V)

As(III)

0 mM

134 mM

668 mM

134 mM

668 mM

381 – – – – –

240 141 5 10 1 1

164 217 4 10 1 –

367 14 6 8 2 5

236 145 7 13 2 –

gels. The spots obtained in A. tenuis leaf proteins revealed distinct patterns for As(III) and As(V) treatments. The well-resolved spots detected in each arsenic treatment are presented in Table 1. The number of total spots was reduced respectively by 37% (134 mM) and by 57% (668 mM) in As(V) treatments and by 4% (134 mM) and by 38% (668 mM) in As(III) treatments compared to controls. Thus, it appeared that more spots thus disappeared with As(V) than with As(III) treatments. Based on the relative volume of each spot, only a few spots showed significant changes with As(V) or As(III) treatments (Table 1). Up-regulated proteins (score > 60 and score < 60) were more abundant than down-regulated ones.

Table 2a Putative identify of A. tenuis proteins totally disappearing after arsenic species treatments. Values are means from three independent experimental replicates. Spot number

A B C D E

Molecular mass (kDa)

56413 – 53445 18403

pI

6.22 – 6.22 8.98

Protein volume Control

Arsenic

0.4 0.93 – 0.89 2.44

0 0 – 0 0

Matching protein

Source

Accession number

Score

%Coverage

RuBisCO RuBisCO – RuBisCO RuBisCO

Agrostis stolonifera – Triticum aestivum Bromus cathaticus

118430310 – 11383 4038725

96 – 130 68

2 – 2 28

LSU LSU LSU SSU

Table 2b Properties and putative identity of A. tenuis proteins affected by arsenic species treatments. Values are means from three independent experimental replicates. Molecular mass (kDa)

pI

Protein spot volume percent Control

As(V) (134 mM)

As(V) (668 mM)

As(III) (134 mM)

As(III) (668 mM)

1 2

16 16

6.68 5.46

3.57  1.18 0.14  0.033

0.476  0.319 0.378  0.0398

0.576  0.123 –

0.655  0.51 –

0.688  0.098 –

3

23

5.26

0.246  0.145

0.464  0.139

0.401  0.179

0.793  0.461

0.530  0.179

4

33

4.75

0.631  0.140

–

8.67  4.22

3.413  1.336

5.592  1.16

5

23

4.81

0.056  0.003

0.235  0.099

0.367  0.114

0.160  0.003

0.2670  0.113

6

23

5.03

0.819  0.589

1.642  1.357

1.127  0.740

2.473  0.446

0.975  0.231

7

33

6.63

1.331  0.392

–

–

0.304  0.015

–

8

32

6.50

0.216  0.026

–

0.765  0.336

0.414  0.099

0.292  0.101

9

43

5.17

0.099  0.032

–

–

1.514  0.765

0.313  0.244

10

37

6.99

0.545  0.061

–

–

–

0.212  0.08

11

17

6.19

0.193  0.101

–

–

–

0.938  0.139

12

27

6.15

0.167  0.08

–

–

–

0.683  0.198

13

43

5.1

0.7  0.3

4.016  1.4539

–

1.023  0.029

1.3  0.1

Matching protein

Source

Accession number

Score

%Coverage

Hypothetical protein Ribulose 1-5 biphosphate carboxylase SSU Ribulose 1.5 biphosphate carboxylase LSU Oxygen-evolving enhancer protein 1 Ribulose 1.5 biphosphate carboxylase/oxygenase LSU Ribulose 1.5 biphosphate carboxylase/oxygenase LSU ribulose 1.5 biphosphate carboxylase LSU (triticum aestivum) Oxygen-evolving protein 2. Chloroplast precursor Ribulose 1.5 biphosphate carboxylase/oxygenase LSU Ribulose 1.5 biphosphate carboxylase/oxygenase LSU Ribulose 1-5 biphosphate carboxylase SSU Ribulose 1.5 biphosphate carboxylase/oxygenase LSU ATP synthase subunit beta

Secale cereale Avena agadiriana Anthoxanthum odoratum Oryza sativa

4090293 6409329

81 123

56 50

57283844

207

53

739292

96

42

Aphyllanthes monspeliens Azorella monantha Triticum aestivum

37722376

81

56

75758063

78

28

12344

118

35

Triticum aestivum Glyceria fluitans Glyceria grandis Avena maroccana Forchhammeria sessilifolia Aegilops columnaris

131394

92

33

41056401

127

33

54303880

175

47

6409329

93

48

46325923

62

51

50401828

139

49

I. Duquesnoy et al. / Plant Science 176 (2009) 206–213

Spot number

209

210

I. Duquesnoy et al. / Plant Science 176 (2009) 206–213

Moreover, the As(III) treatment showed more up-regulated (14.7%) or down-regulated (66.7%) proteins than the As(V) treatment.

(2 spots for 668 mM As(V), 1 spot for 134 mM and 2 spots for 668 mM As(III)).

3.3. Protein identification

4. Discussion

The main protein spots showing significant differences between arsenic treatments and controls were selected for MS analysis. Among these spots, thirty-six spots were excised from preparative 2-D gels and analyzed by MALDI TOF/MS. Table 2a shows the A. tenuis leaf proteins (5) presenting absolute disruption. Table 2b shows A. tenuis leaf proteins (31) with a significant score. Protein spots presenting total disruption during treatment with the two arsenic species were identified as RuBisCO large (LSU) (Acc No. 118430310, spot B; Acc No. 11303, spot D) and small (SSU) (Acc No. 4038725, spot E) subunits (Fig. 2). Protein spots showing the highest up- or down-regulation expression with treatments with the two arsenic species were identified as RuBisCO large (LSU) and small (SSU) subunits, oxygen-evolving enhancer protein 1, oxygenevolving protein 2, and ATP synthase subunit beta. Among the spots with the lowest scores (score < 60) (Table 3), those showing a significant modification in volume were further analyzed. This was the case of proteins matching an Unknown protein (Acc No. 50253074, spot 17), a Hypothetical protein (Acc No. 92876924, spot 23), an Oxygen-evolving enhancer protein, chloroplast precursor (Acc No. 131394, spot 26), a Cysteine protease inhibitor 10 (Acc No. 20137682, spot 16) and an Endogenous alpha amylase subtilisin inhibitor (WASI) (Acc No. 123975, spot 27) whose levels increased with As(V) and As(III). Finally, it is interesting to note that several proteins were found in multiple spots. This was the case of RuBisCO LSU (20 spots, respectively for 134 and 668 mM As(V) and As(III)), RuBisCO SSU (1 spot for 134 mM As(V) and 2 spots for 668 mM As(III)), photosystem II oxygen-evolving complex (PSII-OEC) protein 2 precursor

A. tenuis plants were phenotypically affected by arsenic treatments. The toxicity of arsenic was associated with both the chemical form and the concentration. Application of As(V) did not affect plant growth (fresh and dry biomass), whereas As(III) treatment significantly affected biomass production at the highest concentrations. Moreover, As(III) induced bleaching and necrosis of leaves, while these symptoms were less marked with As(V). Clear necrotic lesions were seen in the first and intermediary leaves of A. tenuis growing in 668 mM As(III). Arsenic toxicity in plants was described by Machlis [29] as consisting of root plasmolysis and leaf wilting followed by root discoloration and necrosis of leaf tips and margins. After 15 days of As(III) treatment, bean plants were wilted, stunted, with foliar chlorosis and necrosis in leaf tips and margins [30]. Our results confirmed that As(III) is more phytotoxic than As(V) [14]. Plant survival has been related to tolerance to arsenic in several cases [31]. Reduction of A. tenuis leaf weight was only detected when plants were grown in the presence of 668 mM As(III). The arsenic found in contaminated water areas did not exceed the concentration of this metal; therefore A. tenuis could grow well in areas with As(V). This observation is in agreement with a previous report by Benson et al. [11] showing that A. tenuis accumulates foliar arsenic concentrations between 2080 and 3470 mg/kg (dry wt.) and is able to grow on As(V) contaminated soils [31]. Carbonell-Barrachina et al. [32] show that As(V) treatment increase dry matter of Spartina patens and Spartina alterniflora. An increase of fresh biomass of P. vittata (hyperaccumulator species) is observed by Tu and Ma [16] after 12 days of As treatment (>334 mM). This

Fig. 2. Two-dimensional IPG  SDS PAGE of Coomassie stained proteins (150 mg loaded) extracted from A. tenuis leaves. A–E spots: RubisCO subunits identified in Table 2a, 1– 31 spots: refers to proteins listed in Tables 2b and 3.

Table 3 Properties and putative identity of A. tenuis proteins affected by arsenic species treatments, with no significant score (<60). Values are means from three independent experiments. Molecular mass (kDa)

pI

As(V) (134 mM)

As(V) (668 mM)

As(III) (134 mM)

As(III) (668 mM)

14

13

6.64

1.33  0.354

3.219  0.959

3.436  1.667

2.045  0.880

2.875  0.684

15 16

23 17

5.26 6.31

0.4425  0.128 0.153  0.057

0.902  0.198 0.564  0.237

0.303  0.098 –

– 0.646  0.170

17 18

43 37

5.10 4.92

0.031  0.008 0.189  0.072

1.272  0.589 0.420  0.146

2.195  1.383 0.512  0.444

0.389  0.011 0.137  0.027

0.225  0.009 0.478  0.216

19 20 21

17 22 42

6.89 4.62 5.03

0.077  0.014 0.042  0.110 0.107  0.053

0.212  0.163 – –

0.477  0.091 0.287  0.023 0.393  0.235

0.154  0.076 0.527  0.217 0.633  0.0157

– 0.400  0.382 0.218  0.149

22 23 24

31 37 20

6.58 4.73 5.45

0.386  0.0689 0.630  0.002 0.866  0.021

0.17  0.013 3.413  1.336 0.464  0.216

– 5.591  1.160 1.182  0.342

25 26

30 25

5.03 6.46

0.006  0.1 0.091  0.073

0.294  0.177 –

0.034  1.3 –

– 5.202  0.57

27

20

5.46

0.241  0.053

0.196  0.112

0.662  0.237

0.958  0.176

28 29

22 30

5.6 5.31

0.158  0.045 0.130  0.043

0.250  0.051 0.234  0.138

0.089  0.009 0.255  0.121

0.420  0.084 0.679  0.349

30

15

6.23

0.289  0.118

31

22

4.62

0.137  0.034

Protein spot volume percent Control

– – –

– 0.559  0.415

– –

– 8.67  4.228 – – 6.116  2.599

– 0.390  0.072 – – 0.429  0.433

–

0.634  0.205

–

0.504  0.227

Matching protein

Source

Accession number

Score

%Coverage

Photosystem I subunit VII 9 kDa polypeptide Hypothetical protein Cyste´ine prote´ase inhibitor 10 Unknown protein Unknown protein

Anthoceros formosae

33301504

52

74

Medicago truncatula Solanum tuberosum

87162636 20137682

44 39

50 30

Oryza sativa Acetabularia acetabulum Oryza sativa Oryza sativa Hordeum vulgare

50253074 3928889

24 33

23 23

51535511 78708268 167096

25 35 51

19 27 29

Medicago truncatula Medicago truncatula Glycine max

78708268 92876924 7263127

35 23 34

72 32 33

Pinus Radiata Triticum aestivum

2935523 131394

31 54

30 30

Triticum aestivum

123975

41

33

Raphanus sativus Oxychloe andina

947125 51574141

20 52

37 35

Bromus catharticus

4038725

44

31

Cicer arietinum

9187460

25

25

Unknown protein Hypothetical protein Ribulose 1.5 biphosphae carboxylase activase isoform 1 Hypothetical protein Hypothetical protein Disease resistance like protein 21 kDa protein precursor Oxygen-evolving enhancer protein. Chloroplast precursor Endogenous alpha amylase subtilisin inhibitor (WASI) Ferredoxin componenet c Ribulose 1.5 biphosphate carboxylase/oxygenase LSU Ribulose 1.5 bophosphate carboxylase/oxygenase SSU Putative 14 kDa proline rich protein

I. Duquesnoy et al. / Plant Science 176 (2009) 206–213

Spot number

211

212

I. Duquesnoy et al. / Plant Science 176 (2009) 206–213

stimulation of biomass could be link to the phosphate uptake. As(V) and phosphate are chemical analogues and are been taken up by the same carrier system in plants. The competition between phosphate and As(V) for uptake would be the basis for the protective effect of P. This could be one advantage of using this grass in phytoextraction protocols. In maize, several proteins, mainly related to oxidative stress responses, have been detected in roots [20]. A clear modification in the expression of proteins involved in primary metabolism, signaling pathways and protein biosynthesis (cysteine synthase, malate dehydrogenase, protein kinase C inhibitor, ATP synthase, putative guanine binding protein) [21] has been observed in shoots. The results of the present study differ considerably from the results of the above works. First, maize is not tolerant to arsenic and hence, the responses triggered by this metalloid would not be expected to be the same as those displayed by A. tenuis. Second, only early responses were analyzed in maize, since the plants were only treated for 24 h. In the proteomic study described here, we analyzed protein expression after 8 days of arsenic treatment. With a same period of 7 days, the rice exposed to sodium arsenate [33] show a large number of transcription factors, stress proteins, and transporters in roots that were not observed in our study. About 381 well-resolved polypeptide spots were quantified in control conditions. After arsenic treatments, most of them were affected by exposure of the plant to arsenic, and 240 and 367 spots were revealed for As(V) and As(III) at 134 mM respectively, and 164 and 236 spots for As(V) and As(III) at 668 mM respectively. For As(V), 37% and 57% of the Coomassie stained spots disappeared after 8 days of 134 and 668 mM treatment respectively. Moreover, the number of polypeptide spots regulated by arsenic treatments was higher at the highest arsenic concentrations used. Quantitative analysis of the protein spots showed that As(III) induced more changes than As(V). Major difficulties were encountered in trying to identify A. tenuis proteins because few genomic and proteomic sequences are available on this genus in databases. While nearly 6084 protein sequences are available on the NCBI database for wheat, which belongs to the same sub family of Poacea, only 235 protein accessions are available in databases for A. capillaries and A. stolonifera and none for A. tenuis. Therefore, several proteins analyzed in this study could not be assigned with certainty since they shared low sequence identity with plant proteins deposited in databases. Polypeptide spots recognized in this study corresponded to conserved proteins involved in photosyntheticenergetic metabolism such as RuBisCO LSU and SSU, oxygenevolving complex protein 1 and 2, and ATPase subunits. Most of these proteins were accumulated during As(III) and As(V) treatments. After arsenic treatments, four more RuBisCO LSU and SSU spots disappeared than in controls. Moreover different proteins identified in this study (RuBisCO LSU and SSU and PSII-OEC protein 2) were detected in multiple spots with different pIs and molecular masses. Total disruption could be due to protease activity on the RuBisCO protein pool. This complex was catabolized to increase leaf amino acid contents for de novo peptides synthesis. Protein complexes can be used for pollutant transport and detoxification for example by sequestration in the vacuole, or compartmentation in the membrane structure. Multiple spots for a single protein could be due to several causes. Post-translational modifications are possible since the most common modifications observed in peptide mass fingerprints of many spots are methylation and phosphorylation [34]. Another possible explanation is the existence of different isoforms derived from multigene families [35– 39]. Moreover, chemical modification of proteins during the

preparation of samples could cause proteolytic degradation and induce multiple spots on 2-DE gels. In our study, the energetic and carbon biochemical processes were affected by As(V) and As(III) for a period of 8 days. A large number of proteins were found to be identical to the RuBisCO LSU or were fragments of it, indicating the damaging effect of arsenic on the photosynthetic apparatus, which has been reported in previous studies [40], but never shown with arsenic. Moreover, photosystem II oxygen-evolving complex protein 2 precursor showed multiple spots. Other heavy metals such as cobalt, zinc and strontium, induce expression of this complex after excessive treatments, while cadmium and mercury drastically reduce its expression [39]. PSII protein 2 precursors are located in chloroplast thylakoid membrane, and excess metal is known to reduce photosynthetic efficiency [41]. After As(III) treatments, A. tenuis showed more over-expression of this protein complex than As(V). This over-expression could be considered as a metabolic disturbance in response to As(III) stress but not as a specific tolerant action. The polypeptide spots with the lowest score (score<60) are candidates for further analysis. Among them, a putative alpha amylase subtilisin inhibitor, a cysteine protease inhibitor, and proteins that display homologies to protein sequences of unknown function in Oryza sativa and Medicago. The present approach revealed proteins associated with defense mechanisms of the leaves (such as cysteine protease 10 precursor). These proteins are known to protect the seed starch reserves against invading insect pathogens or pests. They were up-regulated by arsenic treatments. Besides the degradation of photosynthetic system, the catabolism of starch and protein should be controlled. This reorientation of metabolism could serve to limit the energetic lost. Thus, the energetic potential would be used to other metabolic ways as protective or adaptative activity. In conclusion, our study enabled correlation of proteomic and physiological data, i.e. the disruption of photosynthesis with chlorotic symptoms. Besides the catabolism of the photosynthesis complex, other proteins are involved in the metabolic response to the arsenic treatments used in our study, including some protein candidates belonging to the group of proteins with a low score after MS and database interrogation. To identify the A. tenuis response more accurately it would be useful to use the proteomics approach on samples harvested at an early stage after arsenic treatment. Acknowledgements This work was supported by a grant from ADEME (PNETOX). We are grateful to Pr. L. De La Canale for helpful discussions, to C. Chambon for his skilful assistance and to LIMAGRAIN Industry for providing A. tenuis seeds. References [1] J. Abedin, A.A. Meharg, Relative toxicity of As(III) and As(V) on germination and early seedling growth of rice (Oryza sativa L.), Plant Soil 243 (2002) 57–66. [2] K. Francesconi, P. Visoottiviseth, W. Sridokchan, W. Goessler, Arsenic species in an arsenic hyperaccumulating fern, Pityrogramma calomelanos: a potential phytoremediator of arsenic-contaminated, Sci. Total Environ. 284 (2002) 27–35. [3] R. Datta, D. Sarkar, S. Sharma, K. Sand, Arsenic biogeochemistry and human health risk assessment in organo-arsenical pesticide-applied acidic and alkaline soils: an incubation study, Sci. Total Environ. 372 (2006) 39–48. [4] N. Scott, K.M. Hatlelid, N.E. MacKenzie, D.E. Carter, Reactions of arsenic(III) and arsenic(V) species with glutathione, Chem. Res. Toxicol. 6 (1993) 102–106. [5] A.A. Meharg, J. Hartley-Whitaker, Arsenic uptake and metabolism in arsenic resistant and non resistant plant species, New Phytol. 154 (2002) 29–43. [6] M. Quaghebeur, Z. Rengel, The distribution of As(V) and As(III) in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and As(V) and phosphate supply, Plant Physiol. 132 (2003) 1600–1609.

I. Duquesnoy et al. / Plant Science 176 (2009) 206–213 [7] S. Wang, C.N. Mulligan, Natural attenuation for remediation of arsenic contaminated soils and groundwater, J. Hazard. Mater. B138 (2006) 459–470. [8] H.B. Wang, M.H. Wong, C.Y. Lan, A.J.M. Baker, Y.R. Qin, W.S. Shu, G.Z. Chen, Z.H. Ye, Uptake and accumulation of arsenic by 11 Pteris taxa from southern China, Environ. Pollut. 145 (2007) 225–233. [9] L.Q. Ma, K.M. Komar, C. Tu, W. Zhang, Y. Cai, A fern that hyperaccumulates arsenic, Nature 409 (2001) 579. [10] W.H. Zhang, Y. Cai, C. Tu, L.Q. Ma, Arsenic speciation and distribution in an arsenic hyperaccumulating plant, Sci. Total Environ. 300 (2002) 167–177. [11] L.M. Benson, E.K. Porter, P.J. Peterson, Arsenic accumulation, tolerance, and genotypic variation in plant on arsenical mine wastes in South-West England, J. Plant Nutr. 3 (1981) 655–666. [12] B. Rathinasabapathi, S. Wu, S. Sundara, J. Rivoal, M. Srivastava, L.Q. Ma, Arsenic resistance in Pteris vittata L: identification of a cytosolic triosephosphate isomerase based on cDNA expression cloning in Escherichia coli, Plant Mol. Biol. 62 (2006) 845–857. [13] N. Singh, L.Q. Ma, Arsenic speciation, and arsenic and phosphate distribution in arsenic hyperaccumulator Pteris vittata L. and non-hyperaccumulator Pteris ensiformis L., Environ. Pollut. 141 (2006) 238–246. [14] M. Patra, N. Bhowmik, B. Bandopadhyay, A. Sharma, Comparison of mercury, lead and arsenic with respect to genotoxic effects on plant systems and the development of genetic tolerance, Environ. Exp. Bot. 52 (2004) 199–223. [15] S. Nami Kartal, Y. Imamura, Removal of copper, chromium, and arsenic from CCAtreated wood onto chitin and chitosan, Bioresour. Technol. 96 (2005) 389–392. [16] S. Tu, L.Q. Ma, Interactive effects of pH, arsenic and phosphorus on uptake of As and P and growth of the arsenic hyperaccumulator Pteris vittata L. under hydroponic conditions, Environ. Exp. Bot. 50 (2003) 243–251. [17] S. Tu, L.Q. Ma, G.E. MacDonald, B. Bondada, Effects of arsenic species and phosphorus on arsenic absorption, As(V) reduction and thiol formation in excised parts of Pteris vittata L, Environ. Exp. Bot. 51 (2004) 121–131. [18] B.M. Lange, M. Ghassemian, Comprehensive post-genomic data analysis approaches integrating biochemical pathway maps, Phytochemistry 66 (2005) 413–451. [19] M. Rossignol, J.B. Peltier, H.P. Mock, A. Matros, A.M. Maldonado, J.V. Jorrin, Plant proteome analysis: a 2004–2006 update, Proteomics 6 (2006) 5529–5548. [20] R. Requejo, M. Tena, Proteome analysis of maize roots reveals that oxidative stress is a main contributing factor to plant arsenic toxicity, Phytochemistry 66 (2005) 1519–1528. [21] R. Requejo, M. Tena, Maize response to acute arsenic toxicity as revealed by proteome analysis of plant shoots, Proteomics 6 (2006) 156–162. [22] BRGM, Synthe`se des travaux de Recherche et De´veloppement en France (1999– 2004) sur la the´matique Arsenic, Rapport final BRGM/RP-53252-FR. [23] C. Damerval, D. De Vienne, M. Zivy, H. Thiellement, Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins, Electrophoresis 7 (1986) 52–57. [24] D.J. Skylas, J.A. Mackintosh, S.J. Cordwell, D.J. Basseal, B.J. Walsh, J. Harry, C. Blumenthal, L. Copeland, C.W. Wrigley, W. Rathmell, Proteome approach to the characterisation of protein composition in the developing and mature wheatgrain endosperm, J. Cereal Sci. 32 (2000) 169–188.

213

[25] N. Imin, T. Kerim, J.J. Weinman, B.G. Rolfe, Characterisation of rice anther proteins expressed at the young microspore stage, Proteomics 1 (2001) 1149–1161. [26] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [27] V. Neuhoff, N. Arod, D. Taube, W. Enrhardt, Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brillant Blue G-250, Electrophoresis 9 (1988) 255–262. [28] T. Rabilloud, M. Chevallet, in: T. Rabilloud (Ed.), Proteome Research: Two dimensional Gel Electrophoresis and Identification Methods, Springer, Heidelberg, Germany, 2000, pp. 9–31. [29] L. Machlis, Accumulation of arsenic in shoots of sudan grass and bush bean, Plant Physiol. 16 (1941) 521–544. [30] A.A. Carbonell-Barrachina, F. Burlo, A. Burgos-Hernandez, E. Lopez, J. Mataix, The influence of arsenic concentration on arsenic accumulation in tomato and bean plants, Sci. Horticult. 71 (1997) 167–176. [31] E.K. Porter, P.J. Peterson, Arsenic accumulation by plants on mine waster (United Kingdom), Sci. Total Environ. 4 (1975) 365–371. [32] A.A. Carbonell-Barrachina, M.A. Aarabi, R.D. Delaune, R.P. Gambrell, W.H. Patrick, The influence of arsenic chemical form and concentration on Spartina patens and Spartina alterniflora growth and tissue arsenic concentration, Plant Soil 198 (1998) 33–43. [33] G.J. Norton, D.E. Lou-Hing, A.A. Meharg, A.H. Price, Rice–arsenate interactions in hydroponics: whole genome transcriptional analysis, J. Exp. Bot. 59 (2008) 2267– 2276. [34] A.V. Vener, I. Ohad, B. Anderson, Protein phosphorylation and redox sensing in chloroplast thylakoids, Curr. Opin. Plant Biol. 1 (1998) 217–223. [35] S. Hua, S.K. Dube, N.M. Barnette, S.D. Kung, Photosystem II 23 kDa polypeptide of oxygen-evolving complex is encoded by a multigene family in tobacco, Plant Mol. Biol. 18 (1992) 997–999. [36] J. Gorlach, J. Schmid, N. Amrhein, The 33 kDa protein of the oxygen-evolving complex: a multi-gene family in tomato, Plant Cell Physiol. 34 (1993) 497– 501. [37] T. Sasanuma, Characterization of the rbcS multigene family in wheat: subfamily classification, determination of chromosomal location and evolutionary analysis, Mol. Gen. Genet. 265 (2001) 161–171. [38] L. Porubleva, K. Vander Velden, S. Kothari, D.J. Oliver, P.R. Chitnis, The proteome of maize leaves: use of gene sequences and expressed sequence tag data for identification of proteins with peptide mass fingerprints, Electrophoresis 22 (2001) 1724–1738. [39] K. Zhang, Z.S. Qin, J.S. Liu, T. Chen, M.S. Waterman, F. Sun, Haplotype block partitioning and tag SNP selection using genotype data and their applications to association studies, Genome Res. 14 (2004) 908–916. [40] E. Hajduch, G.J. Litherland, H.S. Hundal, Protein kinase B (PKB/Akt)—a key regulator of glucose transport, FEBS Lett. 492 (2001) 199–203. [41] W. Maksymiec, Effect of copper on cellular processes in higher plants, Photosynthetica 34 (1997) 321–342.