The Effect of Silicon on the Leaf Proteome of Rice (Oryza sativa L

Dec 1, 2010 - Departments of Microbiology and Botany, Miami University, Oxford, Ohio ..... base queries, the Peak Erazor software (v 2.01: Lighthouse ...
0 downloads 0 Views 4MB Size
Download PDF
The Effect of Silicon on the Leaf Proteome of Rice (Oryza sativa L.) Plants under Cadmium-Stress Chika C. Nwugo*,† and Alfredo J. Huerta‡ Departments of Microbiology and Botany, Miami University, Oxford, Ohio 45056, United States Received July 12, 2010

The best known silicon (Si)-accumulating plant, rice (Oryza sativa L.), stores most of its Si in leaves, but the importance of Si has been limited to a mechanical role. Our initial studies showed that Siinduced cadmium (Cd) tolerance is mediated by the enhancement of instantaneous water-use-efficiency, carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO), and light-useefficiency in leaves of rice plants. In this study, we investigated changes in the rice leaf proteome in order to identify molecular mechanisms involved in Si-induced Cd tolerance. Our study identified 60 protein spots that were differentially regulated due to Cd and/or Si treatments. Among them, 50 were significantly regulated by Si, including proteins associated with photosynthesis, redox homeostasis, regulation/protein synthesis, pathogen response and chaperone activity. Interestingly, we observed a Si-induced up-regulation of a class III peroxidase and a thaumatin-like protein irrespective of Cd treatment, in addition to a Cd-induced up-regulation of protein disulfide isomerase, a HSP70 homologue, a NADH-ubiquinone oxidoreductase, and a putative phosphogluconate dehydrogenase, especially in the presence of Si. Taken together, our study sheds light on molecular mechanisms involved in Siinduced Cd tolerance in rice leaves and suggests a more active involvement of Si in plant physiological processes than previously proposed. Keywords: Silicon • rice • cadmium-stress • proteins • stress response • photosynthesis

Introduction The productivity and biological efficiency of plants have been shown to be greatly limited by the presence of heavy metals in the soil at toxic levels.1-3 While some heavy metals like zinc (Zn), copper (Cu), and nickel (Ni) are essential plant micronutrients, other heavy metals such as Cd, lead (Pb), and arsenic (As) have no known biological functions in plants but are readily taken up.3 Among the well-known phytotoxic heavy metals in the environment, Cd is of considerable importance due to its high water solubility, mobility, persistence, and toxicity even in minute amounts.4,5 Rice is a staple food source for more than two-thirds of the world’s population and several rice varieties are cultivated in marshy soils making rice a potential entry point for Cd into the biosphere. For instance, high accumulation of Cd in rice grown in polluted soils in Japan in the mid 1950s and 1960s was responsible for the human itai itai disease.6 The toxicity effects of Cd in plants have been extensively studied, although several questions remain unaddressed.1-3 Symptoms of Cd toxicity in plants include growth inhibition and the disruption of physiological processes.7-12 Plants have evolved mechanisms for alleviating and/or tolerating heavy metal stress, including Cd-stress, some of which include: * Corresponding author. Department of Microbiology, Miami University, Oxford, Ohio 45056, USA. E-mail: [email protected]. Tel: 513-529-6321. Fax: 513-529-2431. † Department of Microbiology. ‡ Department of Botany.

518 Journal of Proteome Research 2011, 10, 518–528 Published on Web 12/01/2010

exclusion, compartmentalization, complexation/sequestration by small molecules such as phytochelatins (PCs), and the synthesis of stress-response proteins.2,5 Additionally, Cd toxicity in plants has also been shown to be alleviated by the presence of certain elements such as Zn and Si.13-16 Silicon is not considered to be an essential element for plants, although recent studies have been geared toward revisiting this issue.17,18 Silicon is the second most abundant element in soils, but its availability to plants as silicic acid could be limiting, hence, the application of silicate fertilizers in crop (rice) production.18 Rice is the most effective known Siaccumulating plant, taking up more than 10% of its dry weight as Si and having a Si/Ca ratio of over 17.19 Although the described effects of Si vary among plant species, Si has been shown to improve disease resistance, light interception, wateruse-efficiency (WUE) and photosynthesis, as well as remediate nutrient imbalances in plants.18,20-23 Furthermore, the addition of Si was shown to alleviate Al toxicity in maize (Zea mays L.),24 Zn toxicity in Irish moss (Minuartia verna L.), and Cardaminopsis halleri,25 as well as Cd toxicity in rice.14 Despite advances made in elucidating the importance of Si in plant biology at the whole-plant level, information on the molecular mechanisms involved in Si-induced stress tolerance in plants is very limited. Our initial studies showed that Siinduced inhibition of Cd toxicity is mediated by an enhancement of instantaneous water-use-efficiency, carboxylation efficiency of RuBisCO, and light-use-efficiency in leaves of rice plants, suggesting that Si is involved in the regulation of 10.1021/pr100716h

 2011 American Chemical Society

Effect of Silicon on the Proteome of Cadmium-Stressed Rice 11,12

physiological processes. Furthermore, studies by Gong et al.26 on wheat and Liang et al.21 on barley reported that Si alleviates abiotic stress by increasing the activities of antioxidant enzymes, which suggests that Si might have an effect on protein expression. The field of proteomics is gaining recognition as a reliable and reproducible high-throughput approach in understanding biological processes.27,28 Proteomics studies involve the systematic analysis and documentation of expressed proteins of a given organism or specific tissue at a given time, and is often regarded as the study of gene expression at the functional level. In this study, we employed a proteomic approach involving 2-DE and mass spectrometry techniques to analyze the protein profiles of the leaf lamina of rice seedlings exposed to Cd and/ or Si treatments. The aim of this study was to identify molecular mechanisms that could be associated with our initial wholeplant studies, which showed that Si-induced alleviation of Cd toxicity involves the regulation of physiological processes in leaves of rice plants. To our knowledge, this is the first study to apply a proteomic approach to investigate the effect of Si on Cd tolerance in rice plants or to plants in general.

Materials and Methods Growth Conditions and Treatemnts. Rice (O. sativa L. var. Jefferson) seeds obtained from USDA-ARS Rice Research Center (Beaumont, TX) were sterilized in 10% bleach and germinated on four layers of sterile paper towels presoaked in doubledistilled H2O. Six days after the beginning of germination, seedlings were transferred to aerated nutrient solutions. Hydroponic plant growth was performed as previously described using 20 µM Fe (III) EDTA.12 The GEOCHEM-EZ chemical speciation program29 was used to predict solution ion activities and interactions. Silicon (0.0 or 0.6 mM) was added as sodium silicate (Na2Si3O7) solution and Cd (0 or 10 µM) was added as Cd sulfate (CdSO4). Silicon and Cd treatments were initiated when seedlings were 6 and 40 days (d) old, respectively. An equivalent amount of Na (as NaCl) was added to the zero Si-treated plants to compensate for the Na content of 0.6 mM Si-treated plants. Treatments were arranged factorially in a randomized experimental design with five replicate seedlings per treatment. Our Si treatment concentration was chosen based on environmental relevance17,19 and because our previous experiments showed that 0.6 mM Si alleviated Cd-induced toxicity symptoms at the whole-plant level.11,12 Furthermore, we chose to use 10 µM Cd rather than 5 µM Cd,12 because plants in the present study had a shorter duration of exposure to Cd treatment. Whole-Plant Physiological Measurements. Gas exchange and chlorophyll fluorescence measurements were commenced on 50 d old plants and were conducted on the third fully expanded leaf from the top using an infrared gas analyzer (LI6400, Licor, Lincoln, NE) and a modulated fluorimeter (OS5FL, Opti-Science, Inc., Tyngsboro, MA), respectively. Measurements of A (instantaneous net CO2 assimilation rate), Amax (maximum net CO2 assimilation rate), gs (instantaneous stomatal conductance rate), gsmax (maximum stomatal conductance rate), Ci (intercellular CO2 concentration), Ca (ambient CO2 concentration in the LI-6400 analyses chamber), CE (carboxylation efficiency of RuBisCO), Fv/Fm (quantum efficiency of open PS2 centers in a dark-adapted state), and qP (photochemical quenching coefficient in a light-adapted state) were conducted as previously described.11 At the end of the growth

research articles

period, total leaf area, total dry weight, tissue-Si, and tissueCd concentrations were determined as previously described.11 Total Protein Extraction. The same leaves that were used during noninvasive physiological measurements were harvested, immediately frozen in liquid nitrogen, and stored at -80 °C for proteomic analysis. The method used for total leaf protein analysis was modified after Tsugita and Kamo.30 Avoiding necrotic regions, 0.4 g of leaf tissue was ground in liquid nitrogen to a fine powder. The powder was transferred to sterile 5 mL polyallomer centrifuge tubes containing 3 mL of chilled solution A [90% (v/v) acetone, 9.9993% (v/v) trichloroacetic acid (TCA), 0.0007% (v/v) Beta-mercaptoethanol]. The mixture was incubated overnight (16 h maximum) at -20 °C followed by centrifugation at 4 °C for 18 min at 36 000g. The supernatant was decanted, and the pellet washed three times until the supernatant was clear (not greenish) by adding 3 mL of chilled solution B [98.53% (v/v) acetone, 1 mM polymethylsulfonylfluoride (PMSF), 2 mM EDTA, 0.0007% (v/v) Betamercaptoethanol], incubating at -20 °C for 1 h, and centrifuging at 4 °C for 18 min at 19 000g. The whitish pellet was then vacuum-dried (Savant Instruments, Inc.) to remove any remaining acetone. The pellet was suspended in 0.5 mL of rehydration/isoelectric focusing (IEF) buffer [8 M Urea, 50 mM dithiothreitol (DTT), 4% (w/v) CHAPS, 0.2% (v/v) 3/10 ampholytes, 0.002% (w/v) bromophenol blue] by incubating at room temperature (RT) for 30 min. Insoluble material was removed by centrifugation at 14 000g for 15 min at RT and the protein content of the supernatant was determined with the bicinchoninic acid (BCA) protein quantification assay following the manufacturer’s instructions (Pierce, Rockford, IL). 2-DE Separation and Image Analysis. For first-dimension electrophoresis, 11-cm long pH 4-7 ReadyStrip IPG strips (BioRad, Hercules, CA) were passively rehydrated overnight at RT with IEF buffer containing 200 µg of protein extract. IEF was done using a PROTEAN IEF cell (Bio-Rad) at a current limit of 50 µA per IpG strip at 20 °C, in two steps: 250 V for 10 min; 8 000 V until a total of 35 000 Vh; a holding step of 500 V was applied. Each focused IPG strip was equilibrated by soaking, with mild stirring, in 4 mL of equilibration base buffer 1 (EBB1) [8 M urea, 2% (w/v) sodium dodecyl sulfate (SDS), 50 mM TrisHCl (pH 8.8), 20% (v/v) glycerol, 1% (w/v) DTT] for 10 min, followed by soaking in 4 mL of EBB2 [same content as EBB1 except DTT was replaced with 2.5% (w/v) iodoacetamide (IAA)]. Second-dimension electrophoresis (SDS-PAGE) was performed in 8-16% gradient SDS-polyacrylamide Tris-HCl gels (Criterion precast gels, Bio-Rad) in a twelve-gel cell system (Criterion Dodeca Cell, Bio-Rad). Protein spots were visualized by staining with Coomassie Brilliant Blue (CBB). Stained gels were scanned using the Bio-Rad VersaDoc Imaging System and gel images were analyzed using the PDQuest software package (version 7.3.0, Bio-Rad). Gels were preserved in 0.02% NaN3 at 4 °C until further analysis. All 2-DE analyses were carried out in triplicate. In each treatment, gels were made from plants grown in a single batch, using four independent replicate plants and three technical replications. Gels were captured under uniform settings by a photoimager (Alpha-imager, Alpha Innotech Corp.). Spots from gels were matched so that a given spot had the same number across all gels. A master gel image containing matched spots across all gels was autogenerated. Missing spots were resolved by extensive analysis using the “landmark” tool and spot volumes were normalized according to the total gel image density as suggested by the PDQuest software package. The gels from all four treatments (i.e., -Cd/-Si, -Cd/+Si, Journal of Proteome Research • Vol. 10, No. 2, 2011 519

research articles

Nwugo and Huerta

+Cd/-Si, and +Cd/+Si) were compared by constructing four different replicate groups (each replicate group contained the gel images corresponding to a specific treatment). In each group, an average quantity was determined for each spot and pairwise quantitative and statistical analysis sets were generated by comparing the volume of a given spot across all treatments. Only spots that had g10-fold increase over background and were present in at least two of the three analytical replicates per plant as well as showed >1.5-fold change (P < 0.05) compared to at least one other treatment group were further analyzed. Trypsin Digestion and Mass Spectrometry. Protein spots were manually excised, reduced with DTT, alkylated with IAA, and digested with sequence grade trypsin in the presence of ProteaseMAX Surfactant according to the manufacturer’s protocol (Promega). Acetonitrile extraction was used to enhance peptide recovery. Tryptic-digests were analyzed via mass spectrometry analysis (Bruker Ultraflex III MALDI-TOF/TOF Mass Spectrometer, Bruker). The target plate was spotted with 2 µL of a 1:1 (v/v) mixture of tryptic-digest and matrix solution (10 mg/mL R-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN/0.1% TFA).

of the peaks present in the mass spectra were assumed to be positively identified proteins. To gain functional information on identified proteins, homology searches using BLASTP (http: www.ncbi.nlm.nih.gov/BLAST) were employed. Statistical Analysis. All gas exchange, chlorophyll fluorescence, and Cd/Si leaf-concentration data were subjected to analysis of variance (ANOVA) using SigmaPlot software Version 11 (Systat Software, Inc., Point Richmond, CA) and means (n ) 5) were separated using the Fischer’s Least Significant Difference (FLSD) test at >95% confidence interval (P < 0.05). Our initial multivariate two-way ANOVA test showed a significant interaction between Cd and Si on our data, whereby the effect of Si generally depended on the level of Cd and vice versa. Thus, in order to separate the main effects of Cd or Si on protein expression, pairwise comparisons to determine significant differences in spot volumes between treatments were performed on standardized log10 values of protein spot volumes using the Student’s t-test analysis at >95% confidence interval (P < 0.05) as provided by the PDQuest software.

Mass spectra were acquired in reflector positive ion mode with an accelerating voltage of 21 kV over the mass range of 700-3000 Da using 1000 laser shots (summed/averaged) per spectrum. A mixture of ACTH and Angiotensin standards (Sigma) was used for external calibration. Monoisotopic peaks with S/N > 5 were selected as the peptide mass fingerprint (PMF) per spot. When necessary, parent ion spectra (from TOF/ TOF fragmentation) were acquired over a range of 40-3000 Da by averaging 2000 laser shots (summed/averaged) per fragmentation spectrum. Protein Identification via Database Queries. Prior to database queries, the Peak Erazor software (v 2.01: Lighthouse data, Odense, Denmark) was used to process peptide mass fingerprints (PMFs) according to the manufacturer’s instructions and as previously described.31 Briefly, PMFs from individual spots were compared together and background/contaminating peaks were determined as peaks within the 700-900 Da range that were common to three or more spots and peaks within the 901-3000 Da range that were common to six or more spots with a precision tolerance of (600 ppm. The generated list of background peaks was calibrated using the “keratin.lst” list provided by the software package and the calibrated background peak list was used to process (calibrate and eliminate background peaks) PMFs.

To elucidate mechanisms that might be involved in Siinduced Cd tolerance in rice plants, we performed whole-plant physiological measurements in order to confirm Si-induced Cd tolerance in our rice plants as reported in our previous study,12 prior to investigating changes in rice leaf proteins in response to Cd and/or Si treatments.

The MASCOT search engine (Matrix Science, London, U.K.) was used to find matches of the PMF and MS/MS fragmentation spectra against O. sativa entries available in the NCBI nonreduntant database and the NCBI number of sequences that matched to our protein/peptide queries at the moment of Mascot search was recorded. Fixed and variable modifications (Cys carbamidomethylation and Met oxidation, respectively) and one missed cleavage were considered. PMF database search was conducted using a maximum mass tolerance of (150 ppm, while MS/MS ions search were conducted with a mass tolerance of (0.6 Da on the parent and 0.3-0.8 Da on fragments; in all cases, the peptide charge was +1. Decoy search was done automatically by Mascot on randomized database of equal composition and size. Except for a few spots which required MS/MS analysis for positive identification, the peptide mixtures that produced the highest statistically significant (P < 0.05) match scores and accounted for the majority 520

Journal of Proteome Research • Vol. 10, No. 2, 2011

Results and Discussion

Effect of Cd and/or Si on Whole-Plant Physiological Parameters of Rice Plants. Our whole-plant physiological analysis showed that treatment with Cd or Si significantly increased the concentration of Cd or Si, respectively, in roots or leaves of rice seedlings (Figure 1A-D). There was significantly more Cd in roots than leaves (Figure 1A,B) but significantly more Si in leaves than roots (Figure 1C,D). Additionally, Cd treatment did not significantly affect root- or leaf-Si concentration (Figure 1C,D). However, in +Cd plants, the addition of Si resulted in a significant 46% and 40% reduction in root- and leaf-Cd concentration (Figure 1A,B), respectively, as well as a significant 36% increase in total leaf area (Figure 1E) and a slight increase (not significant at P < 0.05) in total dry weight (Figure 1F). We also observed a mild induction of chlorosis in the leaves of Cdtreated plants, especially in the absence of Si (data not shown), which could be an indication of Cd-induced nutrient deficiencies as previously documented.5 Additionally, we observed that treatment with Cd reduced Amax (Figure 1G), CE (Figure 1H), and Fv/Fm (Figure 1I), especially in -Si plants. However, neither Cd nor Si treatments significantly affected Ci (Figure 1J). In +Cd plants, the presence of Si increased Amax by 30% (P < 0.05), CE by 24% (not significant at P < 0.05), and Fv/Fm by 15% (not significant at P < 0.05) (Figure 1G-I). However, the addition of Si significantly reduced gsmax by 65% and 42% in -Cd and +Cd plants (Figure 1K), respectively, regardless of Ci, and without affecting Amax, suggesting an enhancement of instantaneous water-use efficiency by Si. The increase in qP in +Cd/+Si plants compared to plants in other treatments (Figure 1L) suggests a Si-induced increase in light-use efficiency under Cd-stress as previously shown.11,12 In general, results from our whole-plant physiological analyses were congruent with those from our previous studies and for the sake of brevity readers can refer to Nwugo and Huerta11,12 for a detailed discussion on the suggested implications of these results. It should also be noted that an

Effect of Silicon on the Proteome of Cadmium-Stressed Rice

research articles

Figure 1. Effects of Si and/or Cd treatments on whole-plant physiological parameters of rice seedlings. Silicon (0 or 0.6 mM) and Cd (0 or 10 µM) treatments were initiated when seedlings were 6 and 40 d old, respectively. Measurements were commenced on 50 d old seedlings. Bars represent means ( SE of five replicate plants per treatment. Statistical significance (P > 0.05) between means was determined using the FLSD test. (A) Concentration of Cd in roots. (B) Concentration of Cd in leaves. (C) Concentration of Si in roots. (D) Concentration of Si in leaves. (E) Total leaf area. (F) Total dry weight. (G) Maximum net CO2 assimilation rate (Amax). (H) Carboxylation efficiency of RuBisCO (CE). (I) Quantum efficiency of open PS2 centers in a dark-adapted state (Fv/Fm). (J) Intercellular CO2 concentration (Ci). (K) Maximum stomatal conductance rate (gsmax). (L) Photochemical quenching coefficient in a light-adapted state (qP).

induction of Cd-induced Fe deficiency can be possible in other plant species.31,32 Effects of Cd and/or Si on the Leaf Protein Profile of Rice Plants. A high resolution of 2-DE gel pattern in a pI range between 4 and 7 was found by staining with CBB. Supporting Information Figure S1 shows representative 2-DE gel images of total leaf proteome of rice plants grown under different Cd and/or Si treatments. Using PDQuest gel analysis software, we detected over 600 spots per gel and over 440 reproducible spots

within replicate gels (Table S1). Using MALDI-TOF/TOF-MS analysis, we identified 60 (Figure 2) out of 87 detected protein spots whose volumes changed significantly due to Cd and/or Si treatments. A close-up view of the profiles of identified spots in representative gels from each treatment group is shown in Figure S2 panels A-Q. It is important to mention that in certain instances more than one spot matched to a given protein, which could be due to a combination of factors including multimerism/protein isoforms, maturation state, degradation, Journal of Proteome Research • Vol. 10, No. 2, 2011 521

research articles

Nwugo and Huerta

Figure 2. PDQuest-generated master gel image showing the general spot pattern of matched protein spots from total leaf proteins of rice seedlings grown under -Cd/-Si, -Cd/+Si, +Cd/-Si, or +Cd/+Si treatments. Silicon (0 or 0.6 mM) and Cd (0 or 10 µM) treatments were initiated when seedlings were 6 and 40 d old, respectively. Total leaf protein was isolated from 50 d old plants. A sum of 200 µg of protein was loaded on a pH 4-7 IpG strip and protein spots were visualized by staining with Coomassie Brilliant Blue (CBB). Mr, relative molecular weight; pI, isoelectric point.

Figure 3. Differentially regulated rice leaf proteins due to Cd and/or Si treatments. (A) Functional category distribution of the 60 identified rice leaf proteins that were differentially produced in response Si and/or Cd treatments. (B) Venn diagram with intersections a, b, c and d, showing the number of identified protein spots that were significantly up- (2) or down- (1) regulated in +Cd/-Si plants compared to -Cd/-Si plants (a), -Cd/+Si plants compared to -Cd/-Si plants (b), +Cd/+Si plants compared to +Cd/-Si plants (c), +Cd/+Si plants compared to -Cd/+Si plants (d).

and/or post-translational modifications. This phenomenon has also been reported in several proteomics studies.7,33 The 60 identified protein spots whose volumes changed significantly due to Cd and/or Si treatments matched to 49 proteins/peptides (Table S2) and according to putative physiological functions were mainly grouped into six categories (Figure 3A), namely, (i) CO2 assimilation/photosynthesis-related proteins (30.0%), (ii) redox homeostasis-related proteins (13.3%), (iii) regulation/protein synthesis related proteins (16.7%), (iv) pathogen-response related proteins (6.7%), (v) chaperones (8.3%), and (vi) energy/metabolism-related proteins (18.3%) (Figure 3A). We did not identify any metal transport-related proteins that were differentially regulated by Cd and/or Si. Studies by Hajduch et al.7 and Lee et al.34 on the effect of Cd on the proteome of leaves and leaves/roots of rice plants, respectively, also did not identify any metal transport-related proteins that were differentially regulated by Cd. This might suggest that metal transport-related proteins are not significantly affected by Cd in rice plants. However, there were several 522

Journal of Proteome Research • Vol. 10, No. 2, 2011

unidentified protein spots whose volumes were significantly changed due to Cd treatment (Figure S3). In any case, it is important to mention that transport proteins are usually membrane-bound/hydrophobic and conventional proteomic analyses strategies are often limited by protein solubility. Among the identified protein spots, the volumes of 4 spots significantly changed (2 up-accumulated and 2 down-accumulated) due to the addition of Si to -Cd plants, while the volumes of 47 spots significantly changed (42 up-accumulated and 5 down-accumulated) due to the addition of Si to +Cd plants (Figure 3B). On the other hand, the addition of Cd to -Si plants significantly changed the volumes of 57 identified protein spots (10 up-accumulated and 47 down-accumulated), while the addition of Cd to +Si plants significantly changed the volumes of 24 protein spots (10 up-accumulated and 14 down-accumulated) (Figure 3B). Supplemental Table S3 shows the spot nos. of identified spots whose volumes changed according to treatment comparisons described in Figure 3B. In general, we identified 50 protein spots corresponding to 39

research articles

Effect of Silicon on the Proteome of Cadmium-Stressed Rice Table 1. Identified Rice Leaf Protein Spots That Were Significantly Regulated by Si

a The spot nos. correspond to the nos. given in Figure 2 and Figure S2. Asterisk (*) denotes protein identification confirmed by MS/MS. b Average spot volume per treatment group (n ) 12); column A, -Cd/-Si; column B, -Cd/+Si; column C, +Cd/-Si; column D, +Cd/+Si. Average spot volumes separated by letters to show significant difference are presented in Supplemental Document 1. c Protein function/name was determined by http:/www.ncbi.nlm.nih.gov/BLAST/. d Theoretical molecular mass (Mr) and isoelectric point (pI) were calculated by http:/www.expasy.org/. Observed Mr and pI can be extrapolated from Figure 2. e Mascot score of protein hit/mascot score of decoy. f Number of matched peptide masses with number of unmatched peptide masses in parentheses. The sequences of matched peptides per spot is provided in Supplemental Document 2. g Percent sequence coverage.

proteins (Table 1) whose volumes were significantly changed by Si and 10 protein spots corresponding to 10 proteins (Table 2) whose volumes were not significantly changed by Si. CO2 Assimilation/Photosynthesis. Our study identified a total of 18 protein spots that matched to 11 proteins/peptides associated with CO2 assimilation/photosynthesis processes representing the largest functional category group of differentially expressed proteins due to Cd and/or Si treatments (Figure 3A, Tables 1 and 2). Hajduch et al.7 showed that heavy metals,

including Cd, inhibited the expression of RuBisCO large subunit, RuBisCO activase, and an oxygen evolving enhancer 2 precursor protein in rice leaves. Furthermore, Kieffer et al.35 showed that proteins involved in carbon fixation and photosynthesis were repressed in young poplar leaves due to Cd treatment. Gillet et al.36 showed that exposure of Chlamydomonas reinhardtii to Cd down-regulated photosynthesis-related enzymes: RuBisCO and oxygen evolving complex proteins 2 and 3. The down-regulation of RuBisCO and other photosynthesisJournal of Proteome Research • Vol. 10, No. 2, 2011 523

research articles Table 2. Identified Rice Leaf Protein Spots That Were Not Significantly Regulated by Si

a The spot nos. correspond to the nos. given in Figure 2 and Figure S2. Asterisks (*) denote protein identification confirmed by MS/MS. b Average spot volume per treatment group (n ) 12); column A, -Cd/-Si; column B, -Cd/+Si; column C, +Cd/-Si; column D, +Cd/+Si. Average spot volumes separated by letters to show significant difference are presented in Supplemental Document 1. c Protein function/name was determined by http:/www.ncbi.nlm.nih.gov/BLAST/. d Theoretical molecular mass (Mr) and isoelectric point (pI) were calculated by http:/www.expasy.org/. Observed Mr and pI can be extrapolated from Figure 2. e Mascot score of protein hit/mascot score of decoy. f Number of matched peptide masses with number of unmatched peptide masses in parentheses. The sequences of matched peptides per spot is provided in Supplemental Document 2. g Percent sequence coverage.

related enzymes have also been observed in response to metals other than Cd.37 Furthermore, the reducing energy generated in the light-dependent reactions of photosynthesis is also important in the reduction of sulfate and nitrate, which are necessary for protein biosynthesis. Additionally, the inhibition of Cd-mediated repression of photosynthesis-related proteins by Si supports and could be responsible for our observation of Si-induced alleviation of Cd-mediated inhibition of Amax (Figure 1G), CE (Figure 1H), Fv/Fm (Figure 1I), and qP (Figure 1L). Redox Homeostasis. Proteins involved in redox homeostasis are usually involved in the prevention of oxidative stress, which is induced by reactive oxygen species (ROS). ROS are byproducts of electron transport and redox reactions from metabolic processes such as photosynthesis and respiration. The production of ROS has been shown to be markedly increased under conditions of abiotic stress.38 Cadmium toxicity has been shown to induce the production of ROS, most likely indirectly because the redox potential of Cd is too low to participate in Fenton-like redox reactions.2 However, the effect of Cd on antioxidants or proteins involved in redox homeostasis is complex and appears to be dependent on growth conditions, species, and level/duration of Cd exposure. Our study showed that Cd repressed several protein spots that matched to redox homeostasis-related proteins such as ascorbate peroxidase (Table 1, spot nos. 49 and 73), Cu-Zn 524

Journal of Proteome Research • Vol. 10, No. 2, 2011

Nwugo and Huerta superoxide dismutase (Table 1, spot no. 72), a putative Znbinding oxidoreductase (Table 1, spot no. 79), a putative glycine dehydrogenase (Table 1, spot no. 123), and a putative peptide methionine sulfoxide reductase (Table 1, spot no. 136), especially in the absence of Si. However, we observed an upregulation of 1-cys peroxiredoxin (Table 2, spot no. 111) irrespective of Si addition. A previous study on excised rice leaves also showed that Cd inhibited the activities of redox homeostasis-related proteins such as superoxide dismutase (SOD), ascorbate peroxidase, and glutathione reductase.9 On the other hand, Kieffer et al.39 showed an induction of several peroxidases, reductases, and aldehyde dehydrogenases, but a repression of Cu-Zn superoxide dismutase and a peroxiredoxin in leaves of poplar (Populus tremula L.) plants exposed to 20 µM Cd-stress. Additionally, Shah et al.40 showed that high Cd levels (100-500 µM) induced SOD activity in rice seedlings, while Ahsan et al.41 showed that Cd induced the accumulation of antioxidant and other stress-related proteins in germinating rice seeds. A reduction in antioxidative protein production as observed in this study could be a consequence of Cd-mediated nutrient deficiency; Cd has been observed to cause Fe-deficiency-like changes in the root protein profile of tomato42 and Fedeficiency has also caused similar reductions in antioxidative proteins in Beta vulgaris.43 Additionally, it is not surprising that Zn-requiring redox enzymes such as Cu-Zn SOD and a putative Zn-binding oxidoreductase were inhibited under Cd treatment because Cd has been shown to compete with Zn uptake and potentially induce Zn-deficiency.13 In any case, a possible increase in oxidative stress due to Cd-induced repression of redox homeostasis-related proteins as observed in this study can result in an inhibition of physiological processes such as growth and photosynthesis as observed in our whole-plants analysis (Figure 1). Thus, the ability of Si to increase the production of redox homeostasis-related proteins under Cdstress could be responsible for Si-induced alleviation of Cdmediated inhibition of growth and photosynthesis processes. Studies by Liang et al.21 on barley and Gong et al.26 on wheat reported that Si alleviated abiotic stress by increasing the activities of antioxidant enzymes. Chaperones. Molecular chaperones (e.g., HSPs, chaperonins, peptidyl-prolyl cis-trans isomerases, and protein disulfide isomerases) are proteins involved in protein folding, refolding, assembly, reassembly, degradation, translocation, and suggested to be involved in stress tolerance.28 In this study, we observed that treatment with Cd inhibited the production of peptidyl-prolyl cis-trans isomerase (Table 1, spot no. 17), a putative 60 kDa chaperonin (Table 1, spot no. 42), and a putative chaperonin 21 precursor (Table 1, spot no. 48), particularly in the absence of Si, but induced the production of protein disulfide isomerase (Table 1, spot no. 43) and an endosperm lumenal binding protein (Table 1, spot no. 70), particularly in the presence of Si. A BLASTp search showed that the endosperm lumenal binding protein has a 99% homology to HSP70 of Cucumis sativus and Ricinus communis. As it occurs with redox homeostasis-related proteins, the effect of heavy metals on molecular chaperones is complex. Studies on cell cultures of Arabidopsis thaliana and on cotyledons of Lepidium sativum L. (garden cress) showed a Cd stress-induced up-regulation of molecular chaperones,44,45 while Kieffer et al.39 showed an initial decrease in HSPs and PPI in poplar leaves upon treatment with 20 µM Cd and Durand et al.46 showed a significant decrease in HSP70 and chloroplast chaperonin 21

Effect of Silicon on the Proteome of Cadmium-Stressed Rice in poplar leaves due to Cd treatment. We suggest that more research would be required to fully comprehend the effect of Cd on the regulation of molecular chaperones in plants. Nevertheless, this study clearly showed that the addition of Si to +Cd plants up-regulated molecular chaperones (Table 1). Regulation/Protein Synthesis. Considering the general inhibition of protein expression due to Cd in this study, it is not surprising that proteins involved in regulation/protein synthesis such as 30S ribosomal protein S1 (Table 1, spot no. 20), EF-G (Table 1, spot no. 44), EF-Tu (Table 1, spot nos. 81 and 99), cysteine synthase (Table 1, spot no. 54), S-adenosylmethionine synthetase (Table 1, spot no. 104), and protein tyrosine phosphatase (Table 2, spot no. 34) were markedly repressed by Cd, especially in the absence of Si. However, we observed a Cdinduced up-regulation of a putative protein kinase (Table 1, spot no. 14) and a putative ubiquitin-specific protease 6 (Table 1, spot no. 119), especially in the absence of Si, all pointing to the fact that Si alleviated Cd-induced toxicity effects on regulation/protein synthesis-related proteins. Similar to our results, Basile et al.47 showed that Cd repressed PTPase at the transcriptional level in the liverwort Lunularia cruciata while Durand et al.46 showed that Cd repressed Elongation factor 1-γ and S-adenosylmethionine synthetase in poplar cambial proteome. Protein tyrosine phosphatases (PTPases) in conjunction with protein tyrosine kinases is known to regulate cell growth, differentiation, and proliferation by controlling cellular tyrosine phosphorylation.48 During translation, EF-Tu and EF-G are required for the addition of new amino acids to the growing polypeptide chain.49-51 Ubiquitinspecific proteases constitute part of the ubiquitin-proteasome system, an ATP-dependent process involved in regulating cellular protein concentration by degrading “unwanted” usually damaged/misfolded proteins,52,53 a process which might also involve the putative protein kinase that was up-regulated by Cd treatment. The fact that the addition of Si alleviated the Cd-toxicity effects on the above-mentioned regulation/protein synthesis-related proteins in rice plants is interesting, although the actual role(s) Si plays in regulating these proteins would require further research, such as inquiries into possible Si-protein interactions. Furthermore, several studies have suggested that phytochelatins (PCs), which are nontranslated sulfur-rich proteins derived from glutathione, are the most important Cd-detoxification molecules in plants cells.2 Glutathione is synthesized from the ATP-dependent reaction between γ-Glu-Cys and Gly.54,55 Thus, it is interesting that, rather than an up-regulation, we observed a down-regulation of cysteine synthase and glutathione-S-transferase (Table 1, spot nos. 31, 97, and 139) as well as neither did we identify nor observe an up-regulation of glutathione synthetase which is closely involved in PC synthesis.54,55 Thus, it is tempting to assume that the susceptibility of our rice plants to Cd-stress might just be due to its relative inability to adequately activate the PC production machinery especially in the absence of Si. However, considering that there were several unidentified protein spots whose volumes increased due to the addition of Cd to our rice plants (Figure S3), we suggest further research in order to fully understand the effect of Cd on PC synthesis-related proteins in rice plants. Pathogen-Response. Our study showed that Cd-induced the production of a pathogenesis-related thaumatin-like protein (Table 1, spot no. 5), a ribonuclease 3 precursor (Table 1, spot no. 30), and a putative Chitinase (Table 2, spot no. 89),

research articles

especially in the absence of Si. Pathogen-response (PR) proteins are generally known to be responsive to biotic stress; however, recent proteomics and transcriptomic studies suggest that these proteins could be involved in multiple stress-response processes, including heavy metal stress in plants.7,39,46,56,57 Kim et al.57 characterized rice PR-10 protein and found that it is involved in a senescence process, possesses RNase activity, and suggested that it may play a role in the constitutive defense mechanisms of plants against both biotic and abiotic stresses. Kieffer et al.35 showed an accumulation of PR proteins in poplar leaves and roots due to Cd treatment. Fecht-Christoffers et al.56 and Fu ¨hrs et al.58 showed differential regulation of PR proteins, including peroxidases, glucanase, chitinase, and thaumatinlike proteins in cowpea due to Mn-stress. In a review, Ahsan et al.28 surmised that heavy metal stress is more likely to induce PR protein expression as part of a general stress response regulated process in plants since any kind of stress can make plants more vulnerable to other sources of stress. Thus, the fact that Si reduced Cd-induced production of PR proteins while improving tolerance to Cd-stress suggests a modulation of protein expression by Si in order to improve protein-use efficiency during periods of stress in a process that could be linked to the later discussed Si-induced increase in water-use and light-use efficiencies. Energy/Metabolism. We observed a Cd-induced up-regulation of aconitate hydratase (Table 1, spot no. 108), especially in the absence of Si, as well as a Cd-induced up-regulation of a NADH-ubiquinone oxidoreductase (Table 1, spot no. 107) and a putative phosphogluconate dehydrogenase (Table 1, spot no. 116), especially in the presence of Si. Aconitate hydratase catalyzes the reversible isomerization of citrate and isocitrate as part of the tricarboxylic acid (TCA) cycle. The NADHubiquinone oxidoreductase is the first energy-transducting complex in the respiratory electron-transport chain. Phosphogluconate dehydrogenase is an enzyme in the pentose phosphate pathway, which forms ribulose 5-phosphate from 6-phosphogluconate. Durand et al.46 showed and suggested a patterned response to Cd-stress in leaves of young poplar plants, whereby photosynthesis-related proteins are down-regulated while proteins involved in respiration and glucose catabolism are upregulated. Although the mechanisms involved in this patternedresponse are not well understood, Kieffer et al.35 showed a Cdinduced inhibition of photosynthesis-related proteins but an up-regulation of proteins involved in carbohydrate catabolism. They suggested that the slowing down of photosynthetic carbon fixation due to Cd-stress would limit carbon availability, which might cause plants to induce processes involved in carbohydrate catabolism resulting in a remobilization of carbon/energy storage compounds.32 In agreement with the same concept, physiological studies by He et al.59 showed an increase in photosynthesis but a decrease in respiration in leaves of Gingko biloba due to ozone exposure. Furthermore, several enzymes involved in carbohydrate catabolism need to be phosphorylated in order to become active, which together with the earlier discussed ATP-dependent ubiquitin-proteasome system, might explain an up-regulation of a putative protein kinase (Table 1, spot no. 14) under Cd-stress.60 Rice Leaf Proteins That Were Differentially Regulated by Si Treatment without Correlation to Leaf-Cd Concentration. The importance of Si in plants has generally been limited to that of a mechanical role.18 In this study, we observed a significant Si-induced inhibition of Cd accumulation in root and leaf tissues (Figure 1A,B). This might provide a canonical/ Journal of Proteome Research • Vol. 10, No. 2, 2011 525

research articles

Nwugo and Huerta

Figure 4. A schematic representation of proposed effects of Si nutrition on whole-plant and molecular processes in leaves of rice plants under Cd-stress. Note the Si-induced production of Class III peroxidase and a thaumatin-like protein in unstressed plants, which among other observed Si-induced poststress regulatory mechanisms, might be contributory to improved tolerance to Cd toxicity effects on physiological processes in +Si plants compared to -Si plants. Upward or downward arrows indicate increase or decrease, respectively, while arrow width indicates magnitude. Asterisk (*) denotes mixed effect of Cd treatment on molecular chaperones in -Si plants.

mechanical explanation to why the observed Si-induced regulation of several of our identified proteins (Table 1) was well correlated with Si-induced reduction in leaf-Cd concentration. However, we identified 6 protein spots whose regulation by Si did not correlate with Si-induced reduction in leaf-Cd concentration (Table 1). Two of these spots, a class III peroxidase (Table 1, spot no. 90) and a putative thaumatin-like protein (Table 1, spot no. 46), were up-regulated by Si irrespective of Cd treatment, while four other protein spots, nos. 43, 70, 107, and 116, were up-regulated under Cd-stress especially in the presence of Si (Table 1). Peroxidases belonging to the class III are particularly secretory plant peroxidases responsible for several physiological functions including: increasing cell wall rigidity by immobilization of extensin and cross-links of matrix polysaccharides;61 alleviation of oxidative stress;62 defense against pathogen penetration, wounding, and abiotic stress;63,64 and biosynthesis of secondary metabolites.65,66 Silicon has long been suggested to improve cell wall integrity but the mechanisms involved are not well understood.67,68 Thus, an induction of class III peroxidase could be responsible for Si-induced cell wall integrity. Additionally, an induction of thaumatin-like proteins, which are PR proteins, might also be responsible for previous documentation of Si-induced disease resistance in plants.18,69 The roles of molecular chaperones: protein disulfide isomerase (Table 1, spot no. 43) and endosperm lumenal binding protein (Table 1, spot no. 70) in stress response, as well as the roles of NADH-ubiquinone oxidoreductase and phosphogluconate dehydrogenase (Table 1, spot no. 116) in carbohydrate catabolism and carbon recycling have been earlier discussed. Lastly, while RuBisCo-associated proteins were significantly up-regulated by Si under Cd-stress (Table 1), proteins associated with photosystems I (Table 2, spot no. 88) and II (Table 2, spot no. 96) as well as carbonic anhydrase (Table 2, spot no. 134) were not significantly regulated by Si under Cd-stress even though they were repressed under Cd-stress. Our previous whole-plant physiological studies11,12 provided initial evidence in support of a 30-year old “window hypothesis”, which suggested that Si in the form of silica bodies deposited in leaf epidermal cells acts as a “window” that could enhance lightuse efficiency by facilitating the transmission of light to the photosynthetic mesophyll tissue.70 Results from our present proteomics study on the effect of Si on photosystems I and II proteins (PS1 and 2) further support this hypothesis, by 526

Journal of Proteome Research • Vol. 10, No. 2, 2011

suggesting that an enhancement of light transmission rather than an increased production of light harvesting complexes (PS1 and 2) is responsible for Si-induced increase in light-use efficiency under Cd-stress. The ability of Si to structurally modify the inter- and intra-cellular environment of plants is also implicated in Si-induced enhancement of water-use efficiency, whereby a Si-cuticle double layer71 is suggested to be responsible for Si-induced decrease in the rate of transpiration (water loss) without inhibiting photosynthesis as observed in this study and our previous studies.11,12

Conclusions Our study provides initial evidence showing that the role of Si in plants might not be limited to that of a mechanical role but that Si might be actively involved in the regulation of biochemical processes particularly the modulation of protein production. Our study identified 50 protein spots that matched to 39 proteins/peptides that were differentially regulated by Si (Table 1). We propose that the efficiency of Si in the alleviation of Cd-stress in rice plants is facilitated by the ability of Si to structurally enhance plant cells and efficiently modulate protein expression without sacrificing fitness. This could make Sitreated plants mechanical sturdier and biochemically more apt to tolerate biotic and abiotic stress even under unstressed conditions. (Figure 4). In general, results from this study provide insights into post-transcriptional regulatory mechanisms induced by Si in plants under abiotic stress conditions. Further studies to determine the effect of Si-induced stress tolerance in roots, the sequential influence of Si on gene/ protein expression especially during periods of stress, and possible Si-protein interactions are currently being explored.

Acknowledgment. This work was supported by funds from the Academic Challenge Award Program of the Botany Department at Miami University, Oxford, OH, and the Sigma Xi Grant-In-Aid of Research Award. We are immensely grateful to Dr. John W. Hawes, of the CBFG Center at Miami University, who recently unexpectedly passed away, for his guidance on 2-DE and mass spectrometry protocols. Supporting Information Available: Figure S1: representative 2-DE gels of total leaf proteome of rice plants. Figure S2: close-up view of differentially expressed protein spots in representative 2-DE gels. Figure S3: Venn diagram with inter-

Effect of Silicon on the Proteome of Cadmium-Stressed Rice sections a, b, c and d, showing the total number of detected protein spots that were significantly up- or down-regulated. Table S1: effects of Cd and/or Si treatments on protein yield, total number of detected spots, total number of consistent spots and total number of up/down-regulated spots. Table S2: list of all identified protein spots grouped according to matched proteins and function. Table S3: Spot nos. of identified spots whose volumes changed according to treatment comparisons described in Fig. 3B. Supplemental document 1: histograms of spot volumes highlighting significant differences. Supplemental document 2: Mascot match results. Supplemental document 3: metal speciation analysis of the growth solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Das, P.; Samantaray, S.; Rout, G. R. Studies on cadmium toxicity in plants: a review. Environ. Pollut. 1998, 98, 29–36. (2) Clemens, S. Toxic metal accumulation, responses to exposure and mechanisms of tolerance in plants. Biochimie 2006, 88, 1707–1719. (3) Kirkham, M. B. Cadmium in plants on polluted soil: Effects of soil factors, hyperaccumulation, and amendments. Geoderma 2006, 137, 19–32. (4) Wagner, J. G. Accumulation of cadmium in crop plants and its consequences to human health. Adv. Agron. 1993, 51, 173–210. (5) Sanita` di Toppi, L.; Gabrielli, R. Response to cadmium in higher plants. Environ. Exp. Bot. 1999, 41, 105-130. (6) Herawati, N.; Suzuki, S.; Hayashi, K.; Rivai, I. F.; Koyama, H. Cadmium, copper, and zinc levels in rice and soil of Japan, Indonesia, and China by soil type. Bull. Environ. Contam. Toxicol. 2000, 64, 33–39. (7) Hajduch, M.; Rakwal, R.; Agrawal, G. K.; Yonekura, M.; Pretova, A. High-resolution two-dimensional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: Drastic reductions/fragmentation of ribulose-1,5-bisphosphate carboxylase/oxygnase and induction of stress-related proteins. Electrophoresis 2001, 22, 2824–2831. (8) Vassilev, A.; Lidon, F. C.; Matos, M. C.; Ramalho, J. C.; Yordanov, I. Photosynthetic performance and content of some nutrients in cadmium- and copper-treated barley plants. J. Plant. Nutr. 2002, 25, 2343–2360. (9) Chien, H.; Wang, J.; Lin, C. C.; Kao, C. H. Cadmium toxicity of rice leaves is mediated through lipid peroxidation. Plant Growth Reg. 2001, 33, 205–213. (10) Souza, J. F.; Dolder, H.; Cortelazzo, A. L. Effect of excess cadmium and zinc ions on roots and shoots of maize seedlings. J. Plant Nutr. 2005, 28, 1923-1931. (11) Nwugo, C. C.; Huerta, A. J. Effects of silicon nutrition on cadmiumuptake, growth and photosynthesis of rice (Oryza sativa L.) seedlings exposed to long-term low level cadmium. Plant Soil 2008, 311, 73–86. (12) Nwugo, C. C.; Huerta, A. J. Silicon-induced cadmium resistance in rice (Oryza sativa L.). J. Plant Nutr. Soil Sci. 2008, 171, 841– 848. (13) Kukier, U.; Chaney, R. L. Growing rice grain with controlled cadmium concentrations. J. Plant Nutr. 2002, 25, 1793-1820. (14) Wang, L.; Wang, W.; Chen, Q.; Cao, W.; Li, M.; Zhang, F. Siliconinduced cadmium tolerance of rice seedlings. J. Plant Nutr. 2000, 23, 1397–1406. (15) Liang, Y. C.; Wong, J. W. C.; Wei, L. Silicon-mediated enhancement of cadmium tolerance in maize (Zea mays L.) grown in cadmium contaminated soil. Chemosphere 2005, 58, 475–483. (16) Shi, X.; Zhang, C.; Wang, H.; Zhang, F. Effect of Si on the distribution of Cd in rice seedlings. Plant Soil 2005, 272, 53–60. (17) Epstein, E. Silicon - Annual Review of Plant Physiology. Plant Mol. Biol. 1999, 50, 641–664. (18) Ma, J. F.; Takahashi, E. Functions of silicon in plant growth. In Soil, Fertilizer, and Plant Silicon Research in Japan, Ed. 1, Elsevier Science Publishers: Amsterdam, (2002); pp 107-108. (19) Ma, J. F.; Takahashi, E. Silicon-accumulating plants in the plant kingdom. In Soil, Fertilizer, and Plant Silicon Research in Japan, Ed. 1; Elsevier Science Publishers: Amsterdam, 2002; pp 63-71. (20) Datnoff, L. E.; Deren, C. W.; Snyder, G. H. Silicon fertilization for disease management of rice in Florida. Crop Prot. 1997, 16, 525– 531. (21) Liang, Y.; Chen, Q.; Liu, Q.; Zhang, W.; Ding, R. Exogenous silicon (Si) increases antioxidant enzyme activity and reduces lipid

(22)

(23)

(24)

(25) (26)

(27) (28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

research articles peroxidation in roots of salt-stressed barley (Hordeum vulgare L.). J. Plant Physiol. 2003, 160, 1157–1164. Gao, X.; Zou, C.; Wang, L.; Zhang, F. Silicon decreases transpiration rate and conductance from stomata of maize plants. J. Plant Nutr. 2006, 29, 1637–1647. Romero-Aranda, M. R.; Jurado, O.; Cuartero, J. Silicon alleviates the deleterious salt effect on tomato plant growth by improving plant water status. J. Plant Physiol. 2006, 163, 847–855. Kidd, P. S.; Llugany, M.; Poschenrieder, C.; Gunse, B.; Barcelo, J. The role of root exudates in aluminum resistance and siliconinduced amelioration of aluminum toxicity in three varieties of maize (Zea mays L.). J. Exp. Bot. 2001, 52, 1339–1352. Neumann, D.; Zur Neiden, U. Silicon and heavy metal tolerance of higher plants. Phytochemistry 2001, 56, 685–692. Gong, H.; Zhu, X.; Chen, K.; Wang, S.; Zhang, C. Silicon alleviates oxidative damage of wheat plants in pots under drought. Plant Sci. 2005, 169, 313–321. Rakwal, R.; Agrawal, G. K. Rice proteomics: current status and future perspectives. Electrophoresis 2003, 24, 3378–3389. Ahsan, N.; Renault, J.; Komatsu, S. Recent developments in the application of proteomics to the analysis of plant responses to heavy metals. Proteomics 2009, 9, 2602–2621. Shaff, J. E.; Schultz, B. A.; Craft, E. J.; Clark, R. T.; Kochian, L. V. GEOCHEM-EZ: a chemical speciation program with greater power and flexibility. Plant Soil 2010, 330, 207–214. Tsugita, A.; Kamo, M. 2-D Electrophoresis of plant proteins. 2-D Proteome analysis Protocols. Methods Mol. Biol. 1999, 112, 95– 97. Larbi, A.; Morales, F.; Abadı´a, A.; Gogorcena, Y.; Lucena, J. J.; Abadı´a, J. Effects of Cd and Pb in sugar beet plants grown in nutrient solution: induced Fe deficiency and growth inhibition. Funct. Plant Biol. 2002, 29, 1453–1464. Lo´pez-Milla´n, A.; Sagardoy, R.; Solanas, M.; Abadı´a, A.; Abadı´a, J. Cadmium toxicity in tomato (Lycopersicon esculentum) plants grown in hydroponics. Environ. Exp. Bot. 2009, 65, 376–385. Nwugo, C. C.; Gaddy, J. A.; Zimbler, D. L.; Actis, L. A. Deciphering the iron response in Acinetobacter baumannii: a proteomic approach. J. Proteomics 2011, 74 (1), 44–58. Lee, K.; Bae, D. W.; Kim, S. H.; Han, H. J.; Liu, X.; Park, C. H.; Lim, C. O.; Lee, S. Y.; Chung, W. S. Comparative proteomic analysis of the short-term responses of rice roots and leaves to cadmium. J. Plant Physiol. 2010, 167, 161–168. Kieffer, P.; Dommes, J.; Hoffmann, L.; Hausman, J. F.; et al. Quantitative changes in protein expression of cadmium exposed poplar plants. Proteomics 2008, 8, 2514–2530. Gillet, S.; Decottignies, P.; Chardonnet, S.; Le Mare´chal, P. Cadmium response and redoxin targets in Chlamydomonas reinhardtii: a proteomic approach. Photosynth. Res. 2006, 89, 201– 211. van Keulen, H.; Wei, R.; Cutright, T. J. Arsenate-induced expression of a class III Chitinase in the dwarf sunflower Helianthus annuus. Environ. Exp. Bot. 2008, 63, 281–288. Mittler, R.; Vanderauwera, S.; Gollery, M.; Van Breusegem, F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004, 9, 490–498. Kieffer, P.; Schroder, P.; Dommes, J.; Hoffmann, L.; Renault, J.; Hausman, J. F. Proteomic and enzymatic response of poplar to cadmium stress. Proteomics 2009, 72, 379–396. Shah, K.; Kumar, R. G.; Verna, S.; Dubey, R. S. Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings. Plant Sci. 2001, 161, 1135–1144. Ahsan, N.; Lee, S. H.; Lee, D. G.; Lee, H.; et al. Physiological and protein profiles alternation of germinating rice seedlings exposed to acute cadmium toxicity. C. R. Biol. 2007, 330, 735–746. ´ lvarez, R.; Abadı´a, A.; Abadı´a, J.; Rodrı´guez-Celma, J.; Rella´n-A Lo´pez-Milla´n, A. Changes induced by two levels of cadmium toxicity in the 2-DE protein profile of tomato roots. J. Proteomics 2010, 73, 1694–1706. ´ lvarez, R.; Andaluz, S.; Rodrı´guez-Celma, J.; Wohlgemuth, Rella´n-A ´ lvarez-Ferna´ndez1, A.; Fiehn, O.; Flor Lo´pezG.; Zocchi, G.; A Milla´n, A.; Abadı´a, J. Changes in the proteomic and metabolic profiles of Beta vulgaris root tips in response to iron deficiency and resupply. BMC Plant Biol. 2010, 10, 120. Sarry, J.; Kuhn, L.; Ducruix, C.; Lafaye, A.; Junot, C.; Hugouvieux, V.; Jourdain, A.; Bastien, O.; Fievet, J. B.; et al. The early responses of Arabidopsis thaliana cells to cadmium exposure explored by protein and metabolite profiling analyses. Proteomics 2006, 6, 2180–2198.

Journal of Proteome Research • Vol. 10, No. 2, 2011 527

research articles (45) Gianazza, E.; Wait, R.; Sozzi, A.; Regondi, S.; et al. Growth and protein profile changes in Lepidium sativum L. plantlets exposed to cadmium. Environ. Exp. Bot. 2007, 59, 179–187. (46) Durand, T. C.; Sergeant, K.; Planchon, S.; Carpin, S.; Label, P.; Morabito, D.; Hausman, J. F.; Renaut, J. Acute metal stress in Populus tremula P. alba (717-1B4 genotype): Leaf and cambial proteome changes induced by cadmium. Proteomics 2010, 10, 349– 368. (47) Basile, A.; Alba di Nuzzo, R.; Capasso, C.; Sorbo, S.; Capasso, A.; Carginale, V. Effect of cadmium on gene expression in the liverwort Lunularia cruciata. Gene 2005, 356, 153–159. (48) Kim, J.; Cho, H.; Ryu, S.; Choi, M. Effects of metal ions on the activity of protein tyrosine phosphatase VHR: highly potent and reversible oxidative inactivation by Cu2+ ion. Arch. Biochem. Biophys. 2000, 382, 72–80. (49) Agrawal, R. K.; Heagle, A. B.; Penczek, P.; Grassucci, R. A.; Frank, J. EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome. Nat. Struct. Biol. 1999, 6, 643–647. (50) Conn, G. L.; Draper, D. E.; Lattman, E. E.; Gittis, A. G. Crystal structure of a conserved ribosomal protein-RNA complex. Science 1999, 284, 1171–1174. (51) Correll, C. C.; Munishkin, A.; Chan, Y. L.; Ren, Z.; Wool, I. G.; Steitz, T. A. Crystal structure of the ribosomal RNA domain essential for binding elongation factors. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 13436–13441. (52) Hochstrasser, M. Ubiquitin-dependent protein degradation. Annu. Rev. Genet. 1996, 30, 405–439. (53) Wilkinson, K. D. Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 1997, 11, 1245–1256. (54) Flocco, C. G.; Lindblom, S. D.; Smits, E. A. H. P. Overexpression of enzymes involved in glutathione synthesis enhances tolerance to organic pollutants in Brassica juncea. Int. J. Phytorem. 2004, 6, 289–304. (55) Innocenti, G.; Pucciariello, C.; Le Gleuher, M.; Hopkins, J.; de Stefano, M.; Delledonne, M.; Puppo, A.; Baudouin, E.; Frendo, P. Glutathione synthesis is regulated by nitric oxide in Medicago truncatula roots. Planta 2007, 225, 1597–602. (56) Fecht-Christoffers, M. M.; Braun, H. P.; Lemaitre-Guillier, C.; VanDorsselaer, A.; et al. Effect of manganese toxicity on the proteome of the leaf apoplast in cowpea. Plant Physiol. 2003, 133, 1935–1946. (57) Kim, S. T.; Yu, S.; Kang, Y. H.; Kim, S. G.; et al. The rice pathogenrelated protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity. Plant Cell Rep. 2008, 27, 593–603.

528

Journal of Proteome Research • Vol. 10, No. 2, 2011

Nwugo and Huerta (58) Fu ¨ hrs, H.; Hartwig, M.; Molina, L. E.; Heintz, D.; et al. Early manganese-toxicity response in Vigna unguiculata L.sa proteomic and transcriptomic study. Proteomics 2008, 8, 149–159. (59) He, X. Y.; Fu, S. L.; Chen, W.; Zhao, T. H.; et al. Changes in effects of ozone exposure on growth, photosynthesis, and respiration of Ginkgo biloba in Shenyang urban area. Photosynthetica 2007, 45, 555–561. (60) Polit, J. T. Protein phosphorylation in Vicia faba root meristem cells during the first steps of leaving principal control points after sucrose application. Plant Cell Rep. 2007, 28, 165–173. (61) Iiyama, K.; Lam, T. B. T.; Stone, B. A. Covalent cross links in the cell wall. Plant Physiol. 1994, 104, 315–320. (62) Passardi, F.; Longet, D.; Penel, C.; Dunand, C. The class III peroxidase multigenic family in rice and its evolution in land plants. Phytochemistry 2004, 65, 1879–1893. (63) Bolwell, G. P.; Bindschedler, L. V.; Blee, K. A.; Butt, V. S.; Davies, D. R.; Gardner, S. L.; Gerrish, C.; Minibayeva, F. The apoplastic oxidative burst in response to biotic stress in plants: a threecomponent system. J. Expt. Bot. 2002, 53, 1367–1376. (64) Ros Barcelo`, A.; Pomar, F.; Lo´pez-Serrano, M.; Pedren ˜ o, M. A. Peroxidase: a multifunctional enzyme in grapevines. Funct. Plant Biol. 2003, 30, 577–591. (65) Pomar, F.; Caballero, N.; Pedren ˜ o, M. A.; Ros Barcelo`, A. H2O2 generation during the auto-oxidation of coniferyl alcohol drives the oxidase activity of a highly conserved class III peroxidase involved in lignin biosynthesis. FEBS Lett. 2002, 529, 198–202. (66) Sottomayor, M.; Ros Barcelo`, A. Peroxidase from Catharanthus roseus (L.) G. Don and the biosynthesis of alpha-3,4-anhydrovinblastine: a specific role for a multifunctional enzyme. Protoplasma 2003, 222, 97–105. (67) Hossain, M. T.; Ryuji, M.; Soga, K.; Wakabayashi, K.; Kamisaka, S.; Fuji, S.; Yamamoto, R.; Hoson, T. Growth promotion and increase in cell wall extensibility by silicon in rice and some Poaceae seedlings. J. Plant Res. 2002, 115, 23–27. (68) Currie, H. A.; Perry, C. C. Silica in Plants: Biological, Biochemical and Chemical Studies. Ann. Bot. 2007, 100, 1383–1389. (69) Fauteux, F.; Re´mus-Borel, W.; Menzies, J. G.; Be´langer, R. R. Silicon and plant disease resistance against pathogenic fungi. FEMS Microbiol. Lett. 2005, 249, 1–6. (70) Kaufman, P. B.; Takeoka, Y.; Carlson, T. J.; Bigelow, W. C.; Jones, J. D.; Moore, P. H.; Ghosheh, N. S. Studies on silica deposition in sugarcane (Saccharum spp.) using scanning electron microscopy, energy-dispersive X-ray analysis, neutron activation analysis and light microscopy. Phytomorphology 1979, 29, 185–193. (71) Yoshida, S.; Ohnishi, Y.; Kitagishi, K. Histochemistry of silicon in rice plant III. The presence of cuticle-silica double layer in the epidermal tissue. Soil Sci. Plant Nutri. 1962, 8, 1–5.

PR100716H