Comparative Proteome Analyses Reveal that Nitric Oxide Is an

Article pubs.acs.org/jpr

Comparative Proteome Analyses Reveal that Nitric Oxide Is an Important Signal Molecule in the Response of Rice to Aluminum Toxicity Liming Yang,† Dagang Tian,‡,§ Christopher D. Todd,∥ Yuming Luo,*,† and Xiangyang Hu*,‡ †

School of life sciences, Jiangsu Key Laboratory for Eco-Agriculture Biotechnology around Hongze Lake, Huaiyin Normal University, Huai’an223300,China ‡ Plant Germplasm and Genomics Center, the Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China § Institute of Biotechnology, Fujian Key Laboratory of Genetic Engineering for Agriculture, Fujian Academy of Agricultural Sciences, Fuzhou, 350003,China ∥ Department of Biology, University of Saskatchewan, Saskatoon, Canada S7N 5E2 S Supporting Information *

ABSTRACT: Acidic soils inhibit crop yield and reduce grain quality. One of the major contributing factors to acidic soil is the presence of soluble aluminum (Al3+) ions, but the mechanisms underlying plant responses to Al3+ toxicity remain elusive. Nitric oxide (NO) is an important messenger and participates in various plant physiological responses. Here, we demonstrate that Al3+ induced an increase of NO in rice seedlings; adding exogenous NO alleviated the Al3+ toxicity related to rice growth and photosynthetic capacity, effects that could be reversed by suppressing NO metabolism. Comparative proteomic analyses successfully identified 92 proteins that showed differential expression after Al3+ or NO treatment. In particular, some of the proteins are involved in reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism. Further analyses confirmed that NO treatment reduced Al3+induced ROS and RNS toxicities by increasing the activities and protein expression of antioxidant enzymes, as well as S-nitrosoglutathione reductase (GSNOR). Suppressing GSNOR enzymatic activity aggravated Al3+ damage to rice and increased the accumulation of RNS. NO treatment altered the expression of proteins associated with cell wall synthesis, cell division and cell structure, calcium signaling and defense responses. On the basis of these results, we propose that NO activates multiple pathways that enhance rice adaptation to Al3+ toxicity. Such findings may be applicable to crop engineering to enhance yield and improve stress tolerance. KEYWORDS: aluminum, nitric oxide, S-nitrosoglutathione reductase, reactive nitrogen species, reactive oxygen species

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stress, including aluminum toxicity.8,9 Al ions possess strong affinity for biomembranes, causing the membranes to become rigid, and facilitates the peroxidation of lipids in phospholipid liposomes. The Al-enhanced, iron-mediated peroxidation is the main source of ROS overaccumulation.9 To avoid oxidative damage to plant cells, both enzymatic and nonenzymatic antioxidant systems are activated to efficiently scavenge ROS. Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR), significantly increase after heavy metal stress. Other antioxidant chemicals, including ascorbic acid, tocopherol, glutathione (GSH) and phenolic compounds, also combat heavy metal stress in plants.10,11 Though many mechanisms have been

INTRODUCTION

Aluminum (Al) toxicity is a major contributing factor that inhibits crop production and decreases crop quality in acidic soil worldwide.1,2 At a soil pH lower than 5, aluminum ions (Al3+) are solubilized and suppress root development in plants. Though there are various other ions in the soil, the Al ion is the major cause of phytotoxicity in acidic soils. Al rapidly inhibits root growth and root apex development at very low concentrations by suppressing root elongation and cell division. It also inhibits cell wall synthesis, destroys plasma membrane integrity and disrupts calcium homeostasis and signal transduction pathways.3−5 Additionally, Al stress decreases photosynthesis and the chlorophyll content by interfering with the photosynthetic electron transport of photosynthesis II (PSII) in wheat and sorghum.6,7 Increases in reactive oxygen species (ROS) such as H2O2 and O2− are already well documented during heavy metal © 2013 American Chemical Society

Received: October 15, 2012 Published: January 17, 2013 1316

dx.doi.org/10.1021/pr300971n | J. Proteome Res. 2013, 12, 1316−1330

Journal of Proteome Research

Article

final concentration of 30 μM, and the treatment solution containing SNAP was changed everyday to maintain SNAP activity. For the inhibitor treatments, the rice seedlings were pretreated with the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 50 μM), the NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 10 μM), the feedback nitrate reductase inhibitor glutamine (Gln, 50 mM), or the HO (heme oxygenase, responsible for carbon monoxide generation) inhibitor zinc protoporphyrin-IX (ZnppIX, 50 μM), for 2 h before Al or NO treatment, respectively. L-NAME and cPTIO (both at 100 mM) were dissolved in DMSO as the stock solution; Gln and ZnppIX were directly dissolved in double-distilled H2O before use.

proposed to explain the underlying mechanism of plant response to Al toxicity, the description of plant response to Al stress at a combined proteomic and physiological level is not well defined. Nitric oxide (NO) is a critical signaling molecule that plays a broad role in modulating plant physiological and biochemical functions.12,13 NO treatment can enhance plant tolerance to heavy metal stress caused by cadmium and copper,14,15 NO can also ameliorate Al-induced inhibition of root growth by reducing Al-induced oxidative damage in Cassia tora and Phaseolus vulgaris.16,17 NO can enhance antioxidant enzyme activities to eliminate the overaccumulation of ROS during environmental stress. However, excess NO can also have lethal effects on plant cell viability. For example, NO can interact with superoxide ions to form reactive nitrogen species (RNS) such as the peroxynitrite anion (ONOO−). Peroxynitrite causes oxidative damage and protein modifications such as Tyr nitration and S-nitrosothiols (SNOs).18 Among these RNS, S-nitrosoglutathione (GSNO), which is formed by the S-nitrosylation reaction of NO with GSH, is considered to be physiologically significant in plants because GSNO can act as a mobile reservoir of NO bioactivity.19 The presence of S-nitrosoglutathione reductase (GSNOR) activity, which catalyzes the NADH-dependent reduction of GSNO to GSSG and NH3, has been reported in different plant species. GSNOR controls the GSNO or NO homeostasis during cadmium stress,18,20 cold stress,19 and heat stress21 and regulates cell death in plant cells.22 The mechanism by which NO is stabilized through GSNOR activity during plant response to acid or aluminum toxicity remains unclear. To explore a new strategy for improving the tolerance of rice seedlings to Al toxicity, we first need to understand the mechanism by which rice responds to Al toxicity. In this study, we applied proteomic and physiological approaches to investigate the dynamic protein profiles in rice seedlings under Al stress. Our results demonstrated that NO is an important signal for modulating reactive oxygen species (ROS) and reactive nitrogen species (RNS) metabolism as rice seedlings respond to Al toxicity. As part of this process, NO functions through several mechanisms, such as initiating cell wall synthesis, and activating cell division and cell cycle and defense responses, to contribute to rice tolerance of Al stress. We propose NO activates multiple pathways that enhance rice adaptation to Al3+ toxicity.

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Measurement of Chlorophyll and Carotenoid Contents

Fresh leaves (0.5 g) were ground using a mortar and pestle with a small amount of quartz sand. The samples were then sequentially ground through the addition of 5 mL of 80% acetone until the samples turned white. The samples were transferred to a 25 mL container, and the mortar and pestle were repeatedly washed. The final volume of the samples was adjusted to 25 mL using 80% acetone and then filtered. Chlorophyll and carotenoid contents were measured at the absorbance at 470, 648.6 and 664.2 nm using a spectrophotometer (BioRad, Hercules, CA).23 Analysis of Chlorophyll Fluorescence

Chlorophyll fluorescence was measured using a PAM (pulseamplitude-modulation) Chlorophyll Fluorometer (Heinz-WalzGmbH, Effeltrich, Germany). After 30-min dark adaptation period for the rice leaves, the initial fluorescence yield (F0) was determined using weak modulated irradiation (0.12 μmol m−2 s−1). The maximum chlorophyll fluorescence yield (Fm) and Fv/ Fm were recorded during a saturating photon pulse (4000 μmol m−2 s−1). The PSII-driven electron transport rates (ETRs) were calculated using the following equation: ETR= (Fm′ − Fs)/Fm′ × I × 0.5 × αleaf, where I is the incident photosynthetic photon flux density, 0.5 is the fraction of absorbed quanta that is used by PSII for C3 plants, and α is the leaf absorbance for rice leaves.24 NO Detection

NO production was measured in the rice root tips using a laserscanning confocal microscope with 4,5-diaminoflorescein diacetate (DAF-2 DA) as described previously. 25 H 2 O 2 formation was visualized using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) fluorescence in the root regions as in a previous report.26 In brief, after the different treatments, the rice root were washed twice with distilled H2O to remove traces of the treatment agents, and incubated with DAF-2 DA (10 μM) or H2DCF-DA (15 μM) for 30 min. After incubation, the rice roots were washed with distilled H2O twice. A laser-scanning confocal microscope (Olympus Optical Co. Ltd., Tokyo, Japan) was used for observing the special fluorescence of H2O2 (excitation at 490 nm, emission at 515 nm) and NO (excitation at 488 nm, emission at 515 nm).

MATERIALS AND METHODS

Plant Growth

Rice seeds (Oryza sativa L.) were cleaned and surface-sterilized in a solution of 2% sodium hypochlorite for 15 min, rinsed five times in sterilized water and germinated in plastic trays lined with wet paper towels for 36 h in the dark (23 °C). The seedlings were grown in Hoagland nutrient solution under controlled conditions (25 °C day/30 °C night cycle; 200 μmol photons m−2 s−1 light intensity; relative humidity of 75−80%). When the third leaf was fully expanded after two weeks of culture, they were treated for the experiments.

S-Nitrosothiols (SNOs) Content Measurements

Chemical Treatment

Total SNOs contents were determined using a previously reported method with minor modifications.27 The SNO measurement was based on the reductive decomposition of nitroso species by an iodine/triiodide mixture to release NO, which was subsequently measured using a gas-phase chemiluminescence method and a NO analyzer (Model 410, 2B Technologies, Boulder, CO). SNOs are sensitive to mercuryinduced decomposition, in contrast to nitroso species such as nitrosamine (RNNOs) and nitrosyl hemes. The samples were

For Al stress, aluminum chloride (75 μM) was added to 1/2 Hoagland nutrient solution, with the active free Al3+ in this solution being approximately 16.5 μM calculated using GeoChem-EZ software (GeoChem-EZ software is available from the Kochian Lab at http://www.plantmineralnutrition.net/JonS. htm). For NO treatment, the NO donor S-nitroso-Nacetylpenicillamine, dissolved into double distilled water as a stock soltion (30 mM), was added to the nutrient solution at a 1317



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homogenates were centrifuged (12000× g, 15 min, 4 °C) and the supernatants were added to five volumes of acetone containing 10% (w/v) TCA and 1% (w/v) DTT. The samples were maintained at −20 °C for 4 h and then centrifuged (25000× g, 30 min, 4 °C). The resulting pellets were washed with acetone containing 1% (w/v) DTT at −20 °C for 1 h and then centrifuged, and the wash step was repeated. The final pellets were vacuum-dried and then dissolved in 8 M urea, 20 mM DTT, 4% (w/v) CHAPS and 2% (w/v) ampholyte (pH 3−10). The samples in ampholyte were vortexed thoroughly for 1 h at room temperature and then centrifuged (25000× g, 20 min, 20 °C), and the supernatants were collected for 2D electrophoresis (2DE). Each experiment was repeated three times. Extracted proteins were first separated by isoelectric focusing (IEF) using gel strips to form an immobilized nonlinear pH gradient from 4 to 7 (Immobiline DryStrip, pH 4−7 NL, 17 cm; Bio-Rad, Hercules, CA) and then by SDS−PAGE using 12.5% polyacrylamide gels. The strips were rehydrated for 16 h in 450 μL of dehydration buffer containing 800 μg of total proteins and a trace of bromophenol blue. The strips were focused at 20 °C for a total of 64 kV/h using the PROTEAN IEF system (Bio-Rad, Hercules, CA). After IEF, the strips were equilibrated for 15 min in equilibration buffer (6 M urea, 0.375 M Tris, pH 8.8, 2% (w/v) SDS, 20% (v/v) glycerol and 2% (w/v) DTT). For 2D SDSPAGE, the strips were placed on top of 12.5% (w/v) SDS-PAGE gels. Gel electrophoresis was performed at 25 mA for 5 h. The gels were stained using the colloidal CBB staining method. After staining, the gels were scanned using the GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA) and PDQUEST software (Bio-Rad, Hercules, CA), on the basis of their relative volume. Parameters were optimized as follows: saliency, 2.0; partial threshold, 4; minimum area, 50. Spots were quantified by determining the ratio of the volume of a single spot to the whole set of spots on the gels. The relative volume of each spot was assumed to represent its expression level. A criterion of p < 0.05 was used to define a significant difference when analyzing the parallel spots between different treatments with one-way ANOVA using SPSS software (http://spss.en.softonic.com/). To compensate for subtle differences in sample loading or gel staining/destaining during individual repeat experiments, the volume of each spot was normalized. Each experiment was repeated three biological times.

homogenized in the extract buffer (50 mM HEPES-KOH, pH 7.5, 1 mM DTT, 1 mM EDTA, 7 mM cysteine, 100 μM diethylenetetraminepentaacetic acid) (1:5; w/v), and centrifuged at 3000× g for 10 min. The supernatants were then incubated with 10 mM N-ethylmaleimide (NEM) for 15 min at 4 °C. For each sample, two aliquots were prepared: the first (i) treated with 10 mM sulphanilamide for 15 min at 4 °C to eliminate nitrite and the second (ii) treated with 10 mM sulphanilamide and 7.3 mM HgCl2 for 15 min at 4 °C to eliminate nitrite and SNOs, respectively. These samples were analyzed using the NO analyzer. The data from aliquots (i) and (ii) represent the total SNO concentration. The entire procedure was performed under a red safety light to protect the SNOs from light-dependent decomposition. Antioxidant Enzyme Activity Assay

To determine antioxidant enzyme activities, 5 g of rice seedlings was homogenized to a fine powder with a mortar and pestle under liquid nitrogen. Next, the powder was ground in 30 mL of extraction buffer containing 50 mM sodium phosphate buffer (pH 7.0), 0.2 mM EDTA and 2% polyvinylpolypyrrolidone (PVPP) for 10 min. The homogenates were filtered through two layers of cheesecloth and centrifuged at 4 °C at 15000× g for 15 min. Supernatants were desalted on a Sephadex G-50 column and used for measuring the enzymatic antioxidant activities. The activities of APX and SOD were measured spectrophotometrically by monitoring the change in A290 and A560, respectively.24 GSNOR activity was assayed spectrophotometrically by monitoring the oxidation of NADH at 340 nm at 25 °C. The activity was expressed as nmol NADH consumed per min per mg protein (e340 = 6.22 mM−1 cm−1).27 In-gel Enzyme Activity Staining

Equal amounts of protein from each sample were loaded on to discontinuous PAGE under nondenaturing conditions (4 °C, 4 h, 35 mA). Native-PAGE activity staining was performed on 10% (w/v) acrylamide slab gels and the in-gel activity staining of SOD and APX were detected as previously described.24 In situ H2O2 and O2− Detection

The In-situ detection of H2O2 and O2− were performed using a previously reported method.28 The amount of O2− in the rice leaves was monitored through incubation and infiltration under vacuum for 3 h and 20 min, respectively, in a solution of 2 mM nitroblue tetrazolium (NBT; N6876, Sigma-Aldrich) in 20 mM phosphate buffer (pH 6.1) containing 10 mM NaN3. The reaction was stopped by transferring the seedlings into distilled water. In order to localize the H2O2 that was produced by the leaves, the treated leaves were immersed and infiltrated under vacuum with 1 mg mL−1 of 3,3-diaminobenzidine (DAB, SigmaAldrich) (pH 3.8) for 3 h and cleared by boiling in alcohol (95%) for 5 min. No coloration was observed when the infiltration was carried out in the presence of ascorbic acid (AsA), thus confirming the H2O2 specificity of DAB staining, In both case, the treated leave were rinsed in 80%(v/v) ethanol for 10 min at 70 °C, mounted in lactic acid/phenol/water (1;1;1;v/v), and photographed.29

In-gel Digestion

Protein spots showing significant changes in abundance during the treatments were excised manually from colloidal CBB staining 2DE gels. Protein digestion with trypsin was performed as follows. Individual spots of interest were excised from the 2DE gels using sterile tips and placed in 1.5 mL sterile tubes. Each polyacrylamide spot was destained with 50 mM NH4HCO3 for 1 h at 40 °C, then reduced with 10 mM DTT in 100 mM NH4HCO3 for 1 h at 60 °C and incubated with 40 mM iodoacetamide in 100 mM NH4HCO3 for 30 min. The gel pieces were minced and allowed to dry, and then rehydrated in 12.5 ng/ μL trypsin (sequencing grade; Roche Diagnostics) in 25 mM NH4HCO3 at 37 °C overnight. The trypsin peptides were extracted from the gel grains with 0.1% trifluoroacetic acid in 50% acetonitrile three times. Supernatants were concentrated in a SpeedVac (Savant Instruments Inc., Farmingdale, NY) concentrator to approximately 10 μL and desalted using Zip-Tips (C18 resin, P10, Millipore Corporation, Bedford, MA). Peptides were eluted from the column with 50% acetonitrile/0.1% trifluoroacetic acid. The protein spots that changed more than 1.5-fold

Protein Extraction and 2D Electrophoresis

Protein extraction and 2D separation were performed according to a reported method with minor modifications.24 Approximately 10 to 20 g of the treated leaves was ground in liquid nitrogen and the total soluble protein was extracted at 4 °C in 5 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 20 mM KCl, 13 mM DTT, 2% (v/v) NP-40, 150 mM PMSF and 1% (w/v) PVP. The 1318



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Figure 1. Effects of NO and its metabolic inhibitors on rice seedling growth under Al stress. (A) Effects of different treatments on the growth of rice seedlings exposed to Al stress. Two-week old rice seedlings were treated with 30 μM AlCl3 for 5 days and the rice seedling phenotypes were recorded. For the NO treatment, the seedlings were cocultivated with 30 μM SNAP and 30 μM AlCl3; For the inhibitor treatments, the seedlings were pretreated with 50 μM cPTIO, 10 mM L-NAME or 50 mM Gln for 3 h, followed by NO or AlCl3 treatment. (B and C) Effect of different treatments on (B) rice seedling root and stem length and (C) leaf chlorophyll and carotenoid contents. The two-week old rice seedlings were treated with Al, NO or different inhibitors for 5 days. The root and stem length and the leaf chlorophyll and carotenoids were measured. Data represent the means of five replicate experiments (±SE). Means denoted by different letters show significant differences according to Tukey’s test (P < 0.05).

Gene Expression Analysis by Reverse Transcription-PCR

and passed the Student’s t test (p < 0.05) were selected and identified by mass spectrometry (MS) analysis.

Total RNA was extracted using Qiagen Plant RNA extraction kit (Qiagen, Germany). After the total RNA was extracted, DNAfree total RNA (5 μg) from different treatments was used for first-strand cDNA synthesis in a 20 μL reaction volume containing 2.5 units of avian myeloblastosis virus reverse transcriptase XL (Takara) and 1 μM of oligo(dT) primer. PCR was performed using 2 μL of a 2-fold dilution of the cDNA, 10 pmol of each oligonucleotide primer, and 1 unit of Taq polymerase (Takara) in a 25 μL reaction volume. The primer sequence information is listed in Supplemental Table 1, Supporting Information. To standardize the results, the relative abundance of rice tubulin was determined and used as the internal standard. The cycle number of the PCR reaction was adjusted to obtain a clearly visible band for the sample with the highest transcription level. Each cDNA sample was run at least twice. Aliquots from the PCR were separated on 1.2% agarose gels and visualized using ethidium bromide. The specific amplification products of the expected sizes were observed, and their identities were confirmed by sequencing.

MALDI-TOF/TOF Analysis and Database Searching

The lyophilized peptide samples were dissolved in 0.1% TFA. MS analyses were conducted using a MALDI-TOF/TOF mass spectrometer 4800-plus Proteomics Analyzer (Applied Biosystems, Framingham, MA, US). MS acquisition and processing parameters were reflector positive mode and 800−3500 Da acquisition mass range. The laser frequency was 50 Hz, and 700 laser points were collected for each sample signal. For each sample, 4 to 6 ion peaks with signal-to-noise ratios greater than 100 were selected as precursors for secondary MS analysis; the TOF/TOF signal for each precursor was accumulated with 2000 laser points. The primary and secondary MS data were transferred into Excel files as inputs to search against an NCBI nonredundant database (NCBInr 20101014); the search was restricted to viridiplantae (green plants) using the MASCOT search engine (www.matrixscience.com). The search parameters were set as follows: no restriction of protein molecular weight; one missed trypsin cleavage allowed; cysteine treated by iodoacetamide; and oxidation of methionine. The peptide tolerance was 100 ppm and the MS/MS tolerance was 0.75kD. Protein identifications were validated manually, with at least 4 peptides matching. The keratin contamination was removed and the MOWSE score threshold was greater than 60 (p < 0.05). According to the MASCOT probability analysis, only significant hits were accepted for the identification of the protein sample.

Western Blotting

SDS-PAGE was performed as described by Laemmli using 12% (w/v) polyacrylamide slab gels.30 For Western blot analysis, the protein samples were electroblotted onto polyvinylidene difluoride (PVDF) membranes using a Trans-Blot cell (BioRad). After transfer, the membranes were probed with the appropriate primary antibodies and HRP-conjugated goat antirabbit secondary antibody (Promega, Madison, WI) and the signals were detected using an ECL kit (GE Company, 1319



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Figure 2. Effects of different treatments on rice leaf photosynthesis under Al stress. (A) Images of Fv/Fm (bottom). The white light photos were used as the control before the leaf photosynthesis fluorescence measurement. The pseudocolor code depicted at the bottom of the image ranges from 0 (red) to 1.0 (purple). Plants were treated as described in the legend to Figure 1 legend under 600 μmol m−2 s−1 light intensity and at 25 °C. The experiment was replicated three times with similar results. One representative leaf is shown. (B) Average Fv/Fm values. Fv/Fm was determined with the whole leaf as the area of interest. The data represent the means of five replicate experiments (±SE). Means denoted by different letters show significant differences according to Tukey’s test (P < 0.05). (C) ETRs determined after 1 day of exposure to different treatments. The data are the means of five replicate experiments (±SE).

Figure 3. Effects of different treatments on NO generation in rice seedlings under Al stress. (A) Detection of NO fluorescence using DAF-2DA staining and confocal microscopy. Two-week old rice seedlings were treated with Al3+ and different inhibitors and scavengers as described in the legend to Figure 1 legend. After 12 h of treatment, the rice root tip was loaded with 10 mM DAF-2 DA and NO fluorescence was imaged after 30 min. Representative images demonstrated the localization of NO production in the rice seedling root. Fluorescence, brightfield and overlapping images are shown in the left, middle and right columns, respectively. Bar = 200 μm. (B) Quantification of the NO content after Al and NO treatment. The rice seedlings were treated as described in the legend to Figure 1 legend for 3 days and the NO contents were measured. Means denoted by different letters show significant differences according to Tukey’s test (P < 0.05).

Evansville, IN). The primary antibodies (all obtained from Agrisera Inc., Vännäs, Sweden) were diluted as follows:

polyclonal antibody against plant GR (1:2000), SOD (1:3000), APX (1:1000), GSNOR (1:2000), HO1 (1:1000) and actin 1320



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Figure 4. Dynamic protein changes in the rice leaves responding to Al and NO stress. Treatment with Al and NO were performed as described in the legend to Figure 1 legend. After 1 day and 3 days of treatment, 1 mg of total protein was extracted from the different plants and loaded in each gel. (A) Representative BR-20 stained 2D gel of total protein from the control plants. (B) Enlarged windows (a−e) from panel A of the spot changes in the representative gels from Al- and NO-treated samples.

leaf pigment accumulation (Figure 1C), and enhanced leaf Fv/Fm ratio and ETRs (Figure 2). In contrast, additional L-NAME and Gln treatment obviously aggravated Al-induced damage to rice growth and photosynthetic capability; as the control, neither LNAME, Gln or cPTIO treatment alone affected rice growth (Figures 1 and 2).

(1:3000). The HO1 antibody was prepared by immunizing the rabbit with the synthesized peptides from the N-termination of Arabidopsis HO1 protein (MAYLAPISSSLSIF).

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RESULTS

(1). NO Treatment Mitigates the Al-induced Inhibitory Effect on Rice Seedling Growth and Photosynthetic Capability

(2). Dynamic Protein Profile Changes in Rice Seedlings in Response to Al and NO Treatments

To understand the underlying mechanism of Al stress on rice seedling growth, the dose effect of Al concentration on rice root and stem length was evaluated. Our results indicated that increases of Al concentration between 30 and 100 uM drastically decreased root growth, whereas further increases in Al above 100 uM had a much more limited inhibitory effect. Thus the concentration used for Al treatment was 30 μM for the subsequent experiments to allow for direct comparisons (Supplemental Figure 1, Supporting Information). As shown in Figure 1A and B, Al treatment significantly suppressed rice seedling growth, including root and stem growth. The pigments in the leaves of plants, including chlorophylls and carotenoids, are important in the evaluation of photosynthetic capability and in the evaluation of plant tolerance to environmental stress. Here, we found that Al treatment reduced the content of chlorophylls and carotenoids (Figure 1C). The ratio of Fv/Fm and ETRs in PSII can also directly indicate the leaf’s photosynthetic capacity. Al treatment impaired leaf photosynthesis and led to a reduction in ETR (Figure 2). NO is reported to assuage cadmium stress in Arabidopsis. Therefore, we investigated the role of NO in the response of rice seedlings to Al stress. As shown in Figure 3A, Al stress increased the emission of NO fluorescence in rice root tips indicated with the NO-specific fluorescence probe DAF-2 DA. The total NO content in rice seedlings was also increased by Al treatment (Figure 3B). Al-induced NO accumulation was completely abolished by treatment with the NO scavenger cPTIO. L-NAME and Gln can be used as inhibitors of plant NOS and NR, respectively.26 Pretreatment with L-NAME or Gln also reduced Al-induced NO accumulation (Figure 3). Exogenous NO treatment through the addition of the artificial NO donor SNAP also mitigated the Al-induced inhibiting effect on rice seedlings, including root and stem growth (Figure 1B), increased

To define the role of NO during the rice response to Al stress, we applied a proteomic approach to investigate the changes in the protein profile. Approximately 1000 proteins were reproducibly resolved on each gel. A total of 256 protein spots were reproducibly detected that showed significant changes in response to the Al or NO treatments (p < 0.05) compared to the control, which received no treatment (Figure 4, Supplemental Figure 2, Supporting Information). Overall, 92 out of the 256 differential protein spots were positively identified using MALDI-TOF MS (Table 1, Supplemental Table 2, Supporting Information). The identified proteins were divided into 9 groups based on their biological functions (Figure 5A). The majority of these proteins were sorted to a general energy and metabolism group, followed by antioxidant enzymes, a transcriptional factor group, protein kinases and phosphatases, and cell structure and division proteins. A hierarchical cluster analysis was conducted to categorize the proteins that showed differential expression profiles during Al stress and NO treatment (Figure 5B). As would be expect for the treatments that induce oxidative stresses, we found that the proteins belonging to the antioxidant system were clustered together. In addition, the proteins involved in NO metabolism, including NR and GSNOR, were up-regulated, suggesting their critical roles during rice seedling exposure to Al stress. (3). NO Treatment Enhances Antioxidant Enzyme Activities and Reduces ROS Overaccumulation

Because NO treatment can enhance the protein accumulation of antioxidant enzymes, we investigated whether Al stress induces ROS accumulation and which antioxidant enzymes are involved in ROS scavenging. We first detected ROS accumulation after Al treatment, specifically H2O2 and O2−. As shown in Figure 6A, Al stress induced a large localized accumulation of H2O2 and O2− in 1321



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Table 1. Identification of Proteins in Rice that are Differentially Expressed Greater than 1.5-Fold after Al or NO Treatment using MALDI-MS/MS Analysis ratiog spot no.

NCBI accession no.a

1

gi|223635526

14

gi|75323108

45 60

gi|75328141 gi|158517785

63

gi|75286798

43 34 79

gi|158513192 gi|75144061 gi|122247193

30

gi|231706

2 3

gi|158564096 gi|75327414

5

gi|75125589

36 82

gi|28558165 gi|75288417

91 94

gi|1709758 gi|60389571

6 18 29 32 65

gi|78099188 gi|19860133 gi|114150550 gi|75135599 gi|121332

76 88 89

gi|88909669 gi|538430 gi|57012737

9

gi|82592857

12

gi|158513704

13

gi|82592858

21 23 42 84

gi|158564103 gi|158513335 gi|122222383 gi|75252053

11

gi|75305701

33

gi|206558314

49

gi|75261388

68 78 92

gi|187470917 gi|91207153 gi|73921025

protein name Probable protein phosphatase 2C 19 Calcium and calmodulindependent serine/threonine kinase Mitogen-activated protein kinase 1 Putative serine/threonine-protein phosphatase PP2A-4 cata Probable protein phosphatase 2C 54 Mitogen-activated protein kinase 5 Methylthioribose kinase 2 Isoform MAPK5b of Mitogenactivated protein kinase Cyclin-dependent kinase A-1 E3 ubiquitin-protein ligase SPL11 Protein disulfide isomerase-like 1-2 Ubiquitin-like modifier-activating enzyme 5 26S protease regulatory subunit 7 E3 ubiquitin-protein ligase Os06g0535400 Proteasome subunit alpha type-6 Proteasome subunit alpha type-7-A L-ascorbate

peroxidase 8 Glutathione reductase Peroxidase 1 Thioredoxin reductase NTRB Glutamine synthetase cytosolic isozyme 1-1 Probable L-ascorbate peroxidase 5 superoxide dismutase Probable glutathione S-transferase GSTF1 Probable indole-3-acetic acidamido synthetase GH3.11 Probable indole-3-acetic acidamido synthetase GH3.8 Probable indole-3-acetic acidamido synthetase GH3.2 Auxin response factor 14 Auxin response factor 23 Gibberellin 20 oxidase 2 Auxin-responsive protein IAA1 Heat stress transcription factor A-4d Heat stress transcription factor C-1a Ethylene-responsive transcription factor 1 transcription factor PCF2 MADS-box transcription factor 55 Myb-related protein Myb4, OsMyb4

theo. Mw/ pIb

exp. Mw/ pIc

score

d

protein kinase and phosphatase 71.4/4.65 68.5/4.75 134

p valuef

Al-1d/ control

Al-3d/ control

Al-1d +SNAP/ control


7.8

0.03

1.05

10.87

1.89

2.29

sc.

e

74.3/5.69

60.2/5.63

210

13.7

0.04

1.20

1.00

1.90

2.30

44.8/5.45 35.7/5.04

43.2/5.45 34.6/5.05

83 114

10.6 13.9

0.01 0.01

1.20 1.20

1.70 1.24

1.90 1.21

2.30 1.15

39.3/5.18

39.6/5.12

143

14.2

0.02

1.14

1.13

2.30

1.70

42.9/5.48 48.2/6.18 42.9/5.48

44.2/5.52 47/6.28 30.3/5.15

106 167 109

13.3 11.0 11.4

0.01 0.04 0.03

1.20 1.32 2.18

1.06 1.52 2.68

1.71 1.84 3.86

1.88 1.88 4.26

34.0/6.52 35/6.55 156 protein degradation 75.2/5.20 75.8/5.1 148 57.3/4.69 56.4/4.6 201

13.7

0.02

1.14

1.67

1.83

2.18

6.5 9.9

0.03 0.04

0.96 1.04

0.96 1.19

1.82 1.33

1.66 1.74

45.6/4.60

44.3/4.54

211

11.2

0.00

1.26

1.27

1.80

1.81

47.6/6.03 26.2/5.02

47.2/6.05 27.5/5.11

231 192

9.8 16.5

0.02 0.04

2.17 1.05

1.46 1.06

3.43 1.71

3.35 1.74

29.6/5.37 27.0/6.59

27.5/6.22 27.9/6.85

142 167

21.1 15.2

0.02 0.04

2.50 1.38

0.86 1.15

4.44 1.72

3.89 1.56

antioxidant enzyme 51.1/5.36 44.2/4.71 53.5/6.24 54.2/6.14 35.3/7.03 34.8/6.75 34.6/6.18 37.5/6.31 39.2/5.73 37.6/5.48

129 152 103 177 189

10.5 10.1 12.2 12.4 15.1

0.02 0.03 0.04 0.02 0.02

1.17 1.06 1.24 1.62 2.03

1.71 1.22 1.69 2.30 2.27

1.91 1.42 1.86 4.32 3.04

2.32 1.54 2.24 4.86 3.29

34.7/5.83 15.2/5.71 24.9/5.99

155 129 103

9.1 28.2 9.9

0.03 0.02 0.03

1.20 1.89 1.85

1.37 1.35 1.70

1.57 5.24 4.08

1.97 6.10 2.30

102

10.1

0.03

9.31

8.65

10.01

0.91

94

10.9

0.04

0.81

0.75

1.27

1.51

134

8.1

0.02

1.31

1.32

1.44

1.24

256 105 166 178

8.7 7.9 13.9 19.6

0.03 0.02 0.03 0.04

0.51 0.51 1.20 0.95

0.69 0.74 1.70 0.83

0.85 0.79 1.90 1.90

0.95 0.88 2.30 2.30

106

10.0

0.01

1.20

0.46

1.90

2.30

30.3/5.33 15.2/5.71 24.9/5.85

hormone related 66.7/5.38 66.7/5.37 66.9/5.60 67.9/5.73

68.6/.58 67./5.80

74.0/6.30 73.5/6.48 94.0/6.35 90.4/6.25 42.5/5.73 41.8/5.65 21.5/5.31 22.5/5.36 transcriptional factors 51.1/5.14 52.2/.21 36.8/6.22

36.2/6.29

98

12.4

0.03

1.12

1.45

1.98

2.35

40.1/4.89

42.2/4.96

117

9.9

0.02

1.00

1.00

1.26

1.38

38.5/5.25 27.5/5.19 27.9/6.56

37.5/5.31 28.7/5.28 29.2/6.59

96 99 122

9.9 20.0 11.7

0.02 0.03 0.04

1.33 1.20 2.47

1.50 0.26 4.03

2.50 1.25 4.69

1.83 1.12 4.05

1322



Article

Table 1. continued ratiog spot no.

NCBI accession no.a

protein name

97

gi|73919925

Nuclear transcription factor Y subunit B-3 Heat stress transcription factor B-2a Heat stress transcription factor A-2e

61

gi|75327423

66

gi|75290369

44 46 48 50 51 55 71 93

gi|75294317 gi|147743027 gi|147636834 gi|122234494 gi|147636925 gi|115502169 gi|75289696 gi|60389571

96 15

gi|115505553 gi|148886790

39

gi|158513197

64

gi|148839598

Actin-related protein 4 Cyclin-A1-4 Cyclin-D4-2 Cyclin-D3-2 Cyclin-D5-1 Expansin-B12 Cyclin-D6-1 Chloroplast envelope membrane protein 19 kDa globulin ATP synthase subunit alpha, mitochondrial Late embryogenesis abundant protein 1 Photosystem II D2 protein

4 54 58 98 73

gi|75288633 gi|114152783 gi|110278805 gi|75298023 gi|281312222

Beta-glucosidase 22 Chitinase 12 14-3-3-like protein GF14-D 17.9 kDa class I heat shock protein Isoform 2 of Beta-glucosidase 32

85

gi|75324283

86

gi|122248703

57

gi|122215721

Probable calcium-binding protein CML14 Probable calcium-binding protein CML25/26 Calcineurin B-like protein 10

7

gi|73917677

8 16 17 19 20 24 25

gi|122203376 gi|296439544 gi|152013453 gi|75102800 gi|73619654 gi|147637716 gi|90179750

26

gi|75126164

27 28

gi|115449589 gi|158513175

31 38

gi|75261631 gi|122212221

40 41

gi|75283208 gi|121332

47 52

gi|121343 gi|3913641

53

gi|62900689

Vacuolar cation/proton exchanger 2 Metal transporter Nramp6 Cytokinin dehydrogenase 8 Phytoene dehydrogenase Ferredoxin–nitrite reductase Allene oxide synthase 3 Cyclin-T1-2 Glucose-6-phosphate isomerase, cytosolic B Ammonium transporter 1 member 2 GSNOR uanine nucleotide-binding protein alpha- 1 subunit Zinc transporter 7 Probable cinnamyl alcohol dehydrogenase 3 Putative cyclin-D7-1 Glutamine synthetase cytosolic isozyme 1-2 Glutamine synthetase Fructose-1,6-bisphosphatase, chloroplastic Probable plastid-lipid-associated protein 3

theo. Mw/ pIb

exp. Mw/ pIc

score

d

sc.

e

p valuef

Al-1d/ control

Al-3d/ control



transcriptional factors 19.1/6.31 19.2/6.26 154

12.4

0.03

1.76

1.23

2.32

2.12

32.8/5.19

32.5/5.10

155

10.5

0.01

3.85

3.69

11.84

12.24

40.2/5.64

41.7/5.55

72

10.7

0.02

1.63

2.13

2.76

3.16

9.8 12.4 10.9 10.1

1.20 0.89 0.93 1.20 1.20 1.00 0.99 1.71

1.43 0.77 0.90 1.70 1.70 1.05 1.07 1.52

1.90 1.34 1.45 1.90 1.90 2.32 1.48 1.05

2.30 1.45 0.96 1.14 2.30 3.08 1.25 1.89

cell division and structure 48.4/5.16 46.8/5.24 105 40.5/5.38 42.7/5.32 118 41.6/4.94 42.6/5.05 137 38.3/4.89 40.2/4.96 167 38.7/4.75 37.8/4.84 162 33.0/4.69 30.8/4.51 144 34.1/5.48 34.6/5.58 156 27.0/6.59 27.8/6.55 99

13.8 12.8 15.3

0.03 0.03 0.04 0.04 0.01 0.02 0.03 0.03

21.0/7.48 55.3/5.85

19.7/6.52 55.4/5.75

106 135

18.3 10.9

0.03 0.04

6.56 1.10

4.83 1.47

7.03 1.57

5.65 1.77

35.6/6.01

36.2/6.02

116

12.3

0.03

0.81

0.93

1.51

1.82

211

14.7

0.04

1.07

1.09

1.36

1.49

0.02 0.02 0.03 0.03 0.04

0.92 3.40 1.20 1.20 1.20

0.85 3.90 1.24 1.24 1.70

1.34 5.39 1.44 7.37 1.90

1.46 6.52 1.38 10.50 2.30

39.5/5.34

40.2/5.41 defense related 59.5/4.96 58.5/4.92 33.6/4.63 33.2/4.54 29.2/4.82 30.6/4.88 17.9/5.79 17.2/5.95 56.7/5.56 30.8/5.62 calcium signal 18.6/4.83 18.1/4.78

176 147 157 233 205

10.7 11.7 13.6 26.1 8.3

139

24.9

1.20

0.93

4.98

5.38

16.4/4.46

133

29.5

1.20

1.70

1.90

2.30

29.9/4.87 30.1/4.80 119 energy and metabolism 47.4/4.85 45.8/4.85 114

18.0

1.26

1.28

2.50

2.42

6.3

1.20

1.70

1.90

1.91

16.1/4.55

58.9/5.19 57.6/5.98 64.7/7.95 66.1/6.82 54.4/6.52 71.0/6.52 62.3/6.45

56.4/5.15 56.2/5.95 54.8/5.98 65.2/6.13 53.6/6.44 70.6/6.55 61.3/6.56

88 95 206 213 144 137 106

8.3 7.9 9.0 7.1 7.8 9.9 9.8

1.20 4.81 5.08 1.20 2.27 0.11 0.16

1.32 5.88 7.22 1.70 3.21 0.18 0.20

1.39 6.30 6.57 1.90 12.47 0.16 0.11

1.67 4.90 6.48 2.30 13.23 0.07 0.11

52.2/6.88

54.5/6.91

104

8.5

0.29

0.68

0.88

0.82

40.8/6.78 44.2/6.65

42.4/6.70 42.2/6.61

98 211

14.7 12.7

1.12 1.55

1.45 1.70

2.13 3.48

2.79 1.75

39.7/6.56 38.5/5.90

38.5/6.45 37.8/5.95

134 128

7.8 13.1

1.80 0.97

1.40 0.74

3.10 1.67

3.81 1.50

35.9/5.78 39.2/5.73

36.1/5.88 39.8/5.80

159 144

13.4 12.7

0.71 1.20

0.95 1.70

1.15 1.90

1.55 2.30

46.6/5.97 43.6/5.00

42.9/5.28 37.9/4.68

126 169

9.9 11.3

1.20 1.20

1.23 1.70

1.29 1.90

0.88 2.30

40.0/4.42

34.4/4.12

125

13.9

1.34

1.48

1.83

2.52

1323



Article

Table 1. continued ratiog spot no.

NCBI accession no.a

56

gi|62900682

62 67 69 70 72 74 75

gi|122248828 gi|310947347 gi|12229998 gi|308191447 gi|84028195 gi|75288486 gi|347662395

77

gi|49618879

80 81 83 90 95

gi|117032 gi|75143802 gi|75119480 gi|152032653 gi|46389970

protein name Probable plastid-lipid-associated protein 2 Alcohol dehydrogenase 1 Metal tolerance protein 6 Spermidine synthase 1 Lipoyl synthase 2 Cysteine synthase Aquaporin NIP1-4 PHD finger protein ALFIN-LIKE 6 affeoyl-CoA-O-methyl -transferase (CCOAOMT) Cytochrome c oxidase subunit 2 Anamorsin homologue 2 Heme oxygenase 1, chloroplastic Esterase PIR7B putative cinnamoyl CoA reductase

theo. Mw/ pIb

exp. Mw/ pIc

score

d

sc.

e

p valuef

Al-1d/ control

Al-3d/ control



energy and metabolism 33.9/5.04 29.9/4.65 89

23.2

0.97

1.00

1.90

1.13

40.9/6.20 41.9/5.50 35.1/5.23 41.5/7.54 33.8/5.36 28.4/5.62 29.6/5.47

39.9/6.26 41.4/5.59 34.7/5.37 36/5.72 34.4/5.48 28.8/5.63 30.6/5.47

93 103 177 157 189 98 151

14.6 9.8 13 16.1 15.6 9.2 15.1

1.75 1.20 3.20 1.03 1.20 1.20 1.20

1.38 1.70 8.70 1.12 1.70 1.36 1.70

1.49 1.90 16.91 1.41 1.90 1.90 1.90

1.39 2.30 11.30 1.38 2.30 2.30 2.30

25.8/5.47

29.6/5.47

187

19.2

0.98

1.70

1.27

1.67

29.2/4.95 27.6/4.88 31.9/6.28 28.8/5.85 24.8/6.52

29.8/5.03 28.8/4.90 25.2/5.18 29.6/6.12 25.0/6.38

136 145 211 2103 252

13.5 14.0 14.2 15.3 13.9

1.42 1.00 1.20 1.83 1.63

2.46 1.35 1.46 1.60 1.52

2.88 3.01 1.41 2.47 2.02

3.72 2.74 0.84 2.49 2.61

a

Database accession numbers according to NCBInr. bTheoretical Mw/pI. cExperimental Mw/pI. dMascot search score against the database of NCBInr. eSequences coverage. fProtein spots showed a significant change in abundance(fold change) by a factor >1.5-fold compared to the control analyzed by LSD test. A p-value of

Comparative Proteome Analyses Reveal that Nitric Oxide Is an

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