Root Proteome of Rice Studied by Itraq Provides Integrated Insight Into Aluminum Stress Tolerance Mechanisms in Plants

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JOURNAL OF PROTEOMICS 98 (2014) 189– 205

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Root proteome of rice studied by iTRAQ provides integrated insight into aluminum stress tolerance mechanisms in plants

Zhan Qi Wanga, Xiao Yan Xua, Qiao Qiao Gonga, Chen Xieb, Wei Fana, Jian Li Yanga, Qi Shan Linc, Shao Jian Zhenga,⁎

aState Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China bCenter for Bioinformatics, State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing 100101, China cUAlbany Proteomics Facility, Center for Functional Genomics, University at Albany, Rensselaer, NY 12144, USA

ARTICLE INFO ABSTRACT

Article history: One of the major limitations to crop growth on acid soils is the prevalence of soluble Received 31 August 2013 aluminum ions (Al3+). Rice (Oryza sativa L.) has been reported to be highly Al tolerant; however, Accepted 27 December 2013 large-scale proteomic data of rice in response to Al3+ are still very scanty. Here, we used an Available online 9 January 2014 iTRAQ-based quantitative proteomics approach for comparative analysis of the expression profiles of proteins in rice roots in response to Al3+ at an early phase. A total of 700 distinct Keywords: proteins (homologous proteins grouped together) with >95% confidence were identified. Aluminum stress Among them, 106 proteins were differentially expressed upon Al3+ toxicity in sensitive and Rice root tolerant cultivars. Bioinformatics analysis indicated that glycolysis/gluconeogenesis was the Quantitative proteomics most significantly up-regulated biochemical process in response to excess Al3+.ThemRNA Tolerant levels of eight proteins mapped in the glycolysis/gluconeogenesis were further analyzed by Sensitive qPCR and the expression levels of all the eight genes were higher in tolerant cultivar than in sensitive cultivar, suggesting that these compounds may promote Al tolerance by modulating the production of available energy. Although the exact roles of these putative tolerance proteins remain to be examined, our data lead to a better understanding of the Al tolerance mechanisms in rice plants through the proteomics approach.

Biological significance Aluminum (mainly Al3+) is one of the major limitations to the agricultural productivity on acid soils and causes heavy yield loss every year. Rice has been reported to be highly Al tolerant; however, the mechanisms of rice Al tolerance are still not fully understood. Here, a combined proteomics, bioinformatics and qPCR analysis revealed that Al3+ invasion caused complex proteomic changes in rice roots involving energy, stress and defense, protein turnover, metabolism, signal transduction, transport and intracellular traffic, cell structure, cell growth/division, and transcription. Promotion of the glycolytic/gluconeogenetic pathway in roots appeared crucially important for Al tolerance. These results lead to a

⁎ Corresponding author. Tel./fax: +86 571 88206438. E-mail addresses: [email protected] (Z.Q. Wang), [email protected] (X.Y. Xu), [email protected] (Q.Q. Gong), [email protected] (C. Xie), [email protected] (W. Fan), [email protected] (J.L. Yang), [email protected] (Q.S. Lin), [email protected] (S.J. Zheng).

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190 JOURNAL OF PROTEOMICS 98 (2014) 189– 205

1. Introduction (GWA) and quantitative trait loci (QTL) mapping, 48 QTLs associated with Al3+ tolerance have been identified in rice [24]. Aluminum (mainly Al3+) limits the agricultural productivity Furthermore, several Al-resistant genes have also been cloned on more than one third of the world's total arable and by mutant approaches in rice, for example, ART1 (Al3+ resistance potential arable lands, as the rhizotoxic species Al3+ is transcription factor 1) [4,25], STAR1/STAR2 (sensitive to Al released into soil solutions when the soil pH is below 5.0, rhizotoxicity 1 & 2) [26], Nrat1 (Nramp aluminum transporter 1) accumulating to levels that inhibit root elongation and growth [27], FRDL4 (ferric reductase defective3-like 4) [28], ALS1 (aluminum by damaging the root cells structurally and functionally [1–4]. sensitive 1) [29] and MGT1 (magnesium transporter 1) [30].Allof It has been well demonstrated that the root apex is the most these genes are specifically induced by Al and knockout of any sensitive part of the root to Al3+ because it is the site of cell of them results in the decreasing in Al tolerance, indicating division and cell elongation [5,6]. Since Al3+ is so reactive, it their huge contributions to the high Al tolerance observed in can interact with multiple structures in the apoplast and rice. More recently, through comparative genome-wide tran- symplasm of root cells [3,7]. For instance, in the cell wall, scriptional analysis of wild type and star1 mutant, a novel Al3+ primarily binds to the pectin matrix and thereby alters platform has been provided for further work on Al tolerance in the physical properties of the cell wall [7–9]. In the symplasm, rice [31]. However, the mechanisms underlying high Al toler- sites of Al3+ interaction include membrane constituents, ance in rice are still not well understood at the molecular level. ion channels, metabolic enzymes, components of signaling So, a more thorough analysis at proteome or metabolome levels pathways, members of the cytoskeleton, and even the DNA is required to clarify the mechanisms of Al tolerance in rice. [3,7,10]. Furthermore, Al3+ usually results in an “acid soil Although transcriptomics provides a useful tool for unraveling syndrome” that includes poor plant water and nutrient gene expression networks, proteomics links these networks to uptake, which makes plants sensitive to various stresses, protein products and provides further insight into posttran- especially drought stress [11]. Therefore, Al toxicity has been scriptional modifications that regulate cellular functions, there- recognized as a major factor limiting crop production world- by complementing genomics analysis [32]. In the past two wide on acid soils, particularly in the developing countries. decades, two-dimensional electrophoresis (2-DE) has been In the long-term coevolution, some plant species or cultivars widely used for protein separation and analysis, while iTRAQ have developed two primary strategies to cope with Al toxicity, (isobaric tags for relative and absolute quantification) is a robust which are so-called “internal detoxification mechanisms” and mass spectrometry technology that allows quantitative com- “external detoxification mechanisms” [12,13]. Internal detoxifi- parison of protein abundance by measuring peak intensities of cation is mainly achieved by sequestration of Al3+ into the reporter ions released from iTRAQ-tagged peptides by fragmen- vacuoles in the form of Al–organic acid complexes or redistri- tation during MS/MS [33]. Given iTRAQ's ability to perform bution to the shoots through the symplastic pathway [3],which relative and absolute quantification in up to four or even eight is seen in some Al-accumulating plants such as hydrangea phenotypes, accuracy at quantification, and high-resolution (Hydrangea macrophylla) and buckwheat (Fagopyrum esculentum). precursor ion selection and extended protein and peptide Over the past years, several mechanisms for the external fractionations [34], iTRAQ-based proteomics has been widely detoxification have been proposed, but the most-studied one applied in systems ranging from microorganism stress re- is the secretion of organic acid anions from the roots in sponses [35] to evaluating plants in response to deficient or response to Al stress in both monocots and dicots [3,4,14].The excess nutrient elements [36,37] in the past decade. However, organic acid anions secreted from roots include citrate, oxalate, this technique has not been used to investigate the plant and malate, depending on plant species. All of them are able to responses to Al3+ toxicity. chelate toxic Al3+, thereby detoxify Al3+ in the rhizosphere Here we employed an iTRAQ-based quantitative proteo- [2,3,15]. Recently, genes responsible for Al-induced secretion of mics approach to elucidate the effects of Al3+ toxicity on malate and citrate have been identified, such as ALMT1 in the protein levels in rice cultivars differing in Al tolerance in wheat [16], HvMATE in barley [17] and SbMATE in sorghum [18]. detail. With these measurements, we aimed to identify Transforming these genes into Al-sensitive cultivars has been protein abundance changes in response to Al with the proved to increase Al resistance significantly [19]. potential mechanistic relevance to the early phase of Al3+ Rice (Oryza sativa L.) is an important crop worldwide with a toxicity. We found that glycolysis/gluconeogenesis was the high basal level of tolerance to Al compared with other most significantly up-regulated biochemical process in re- small-grained cereals [20–22]. As a model species, rice is an sponse to excess Al3+. The mRNA levels of eight proteins attractive system for mutational and genetic analyses at both mapped in this pathway were further analyzed by qPCR and gene and protein levels. It has been well demonstrated that the expression levels of all the eight genes were higher in different from those crops which employ secretion of organic tolerant cultivar than in sensitive cultivar, suggesting that acid anions as a main mechanism of Al tolerance, organic acid these compounds may promote Al tolerance by modulating anion secretion is not the major mechanism for high Al the production of available energy and the tolerant cultivar tolerance in rice because the amount of secretion is very small might have a higher potential to produce more energy to [4,21–23]. Recently, through genome-wide association analysis counteract the toxic effect of Al3+. 中国科技论文在线 http://www.paper.edu.cn

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2.3. Trypsin digestion, iTRAQ™ labels, off-line strong cation 2. Materials and methods exchange chromatography and online nano-LC ESI QqTOF MS Analysis off-line strong cation exchange chromatography and 2.1. Plant materials, growth conditions and Al online nano-LC ESI QqTOF MS analysis stress treatments These were performed essentially as described previously Two rice (Oryza sativa L.) cultivars belonging to sp. japonica [43], and are described in details in Supplementary Materials ‘ ’ were used in this study. Nipponbare is the most tolerant to Al and Methods. The general workflow of the iTRAQ experiment – ‘ ’ among the small-grain cereal crops [20 22]. 7098 is a native is shown in Fig. 1. cultivar sensitive to Al, which is gifted by Prof. Jian Feng Ma in Okayama University (Kurashiki, Japan). 2.4. Database search and iTRAQ quantification For relative root elongation (RRE) analysis, seeds of ‘ ’ ‘ ’ Nipponbare and 7098 were soaked in water overnight at MS/MS spectra were processed by a “through” search against a 28 °C in the dark and then transferred to a net floating on JCVI Rice Genome Annotation Release 7 database (132,676 0.5 mM CaCl2 solution. On day 7, seedlings were transferred entries) using the Paragon Algorithm [44] within the to a 1.4-L plastic pot containing Kimura B complete nutrient ProteinPilot v4.0 software (AB SCIEX) with trypsin as the solution [38] which was renewed every 3 days under green- digest agent, cysteine alkylation, an ID focus of biological house conditions (average temperature of 28/24 °C (day/night), modifications, amino acid substitutions, and other default – relative humidity 60 80%, photosynthetically active radiation settings [45]. The software calculates a percent confidence – μ −2 −1 200 400 mol m s and photoperiod of 14 h day/10 h night) which reflects the probability that the hit is a false positive, so [23]. The 4-leaf-old seedlings (approximately three weeks) that at the 95% confidence level there is a false-positive 3+ were then treated with Al as described previously [39,40]. identification rate of 5%. This software automatically accepts Briefly, the seedlings were pre-grown in 0.5 mM CaCl2 solution all peptides with a confidence of identification >1%; therefore, (pH 4.3) overnight for root rinsing and adaptation, and then only proteins that had at least one peptide with >95% transferred into 0.5 mM CaCl2 solution containing different confidence were initially recorded. Simultaneously, the false μ concentrations of AlCl3 (0, 50, 100, 150, 200, 250 or 300 M, discovery rate (FDR) analysis was performed using the 3+ pH 4.3). No addition of Al was used as the control. The RRE Proteomics System Performance Evaluation Pipeline (PSPEP) was defined as the percentage of the root elongated of the software [46] as an add-on to the ProteinPilot software. Using plants receiving Al treatment to that of the no Al control. RRE the ProteinPilot software, quantification was based on the analysis was conducted essentially as described previously signature peak areas (m/z: 113, 114, 115, 116, 117, 118, 119 and [41]. 121) and corrected according to the manufacturer's instruc- For proteomic analysis, the rice seedlings were cultured as tions to account for isotopic overlap. Relative quantification μ has been described above, and 250 M AlCl3 was chosen to ratios of the identified proteins were calculated, averaged and analyze the defensive responses of the roots in response to corrected for any systematic errors in the labeling of the 3+ 3+ – Al . No addition of Al was used as the control. The 2 3cm iTRAQ™ peptides. The accuracy of each protein ratio is given portions from root tips were sampled after the 12 h and 24 h by a calculated “error factor” in the software. The error factor treatments. The root tips of 30 plants were collected as one (EF) expresses the 95% uncertainty range (95% confidence independent biological replicate and three biological repli- error) for a reported ratio, where this 95% confidence error is cates (90 plants) were combined into one sample to give the weighted standard deviation of the weighted average of sufficient sample quantity for one technical repeat. In the log ratios multiplied by the Student's t-factor for n − 1 degrees same way, we got another technical repeat. The two technical of freedom, where n is the number of peptides contributing to repeats were analyzed using an 8-plex iTRAQ experiment relative protein quantification. To be identified as being which had six biological replicates and represented 180 plants significantly differentially expressed, a protein must contain at each treatment time for every rice cultivar. Two indepen- at least two unique high scoring peptides (peptide confidence dent 8-plex iTRAQ experiments were conducted for 12 h and >95%), an error factor of <2 and a ratio fold change >1.1 or <0.9 24 h treatments, respectively. And, they were also termed in both technical replicates. These limits were selected on the experiment I and experiment II, respectively. basis of our previous work with iTRAQ reagents [43,45] with some modifications. 2.2. Root protein extraction and precipitation 2.5. Protein classification and identification Al-responsive Roots (approximately 0.5 g fresh weight) were immediately pathways using KOBAS immersed in liquid nitrogen, then powdered and homoge- nized with RIPA buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, Protein classification was performed essentially with refer- 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 1% sodium ence to the functional categories established by Bevan et al. deoxycholate, 2% SDS) and before using, added one cOmplete [47]. ULTRA Tablet (Roche) for each 10 mL extraction solution. The The software KOBAS 2.0, which stands for Kyoto Encyclo- homogenates were shaken repeatedly for 30 min and centri- pedia of Genes and Genomes (KEGG) Orthology-based Anno- fuged at 20,000g at 4 °C for 40 min, and the supernatants were tation System (http://kobas.cbi.pku.edu.cn) [48], was used subjected to a Methanol/Chloroform precipitation according to identify significant pathways involved in response to Al to Wessel and Flügge [42] with some modifications. at early phase. Because a large number of pathways were 中国科技论文在线 http://www.paper.edu.cn

192 JOURNAL OF PROTEOMICS 98 (2014) 189– 205

Fig. 1 – Strategy for analysis of protein expression in rice roots by 8-plex isobaric tagging. S1, roots of sensitive control plants (7098); S2, roots of sensitive plants (7098) treated with Al3+; T1, roots of tolerant control plants (Nipponbare); T2, roots of tolerant plants (Nipponbare) treated with Al3+.

involved, we implemented FDR correction to control the (Premier Biosoft International, Palo Alto, CA) according to overall Type I error rate of multiple testing using GeneTS mRNA sequences obtained from the Rice Genome Annotation (2.8.0) in the R (2.2.0) statistics software package [49]. Pathways Project and they can be found in Supplementary Table S1. Rice with FDR-corrected p-values <0.05 were considered statistically histoneH3 gene was used as endogenous control which was significant. stable throughout the Al treatment [25]. qPCR was performed in an optical 384-well plate, including 10 μL 2× SYBR Green 2.6. qPCR analysis Master mix reagent (TaKaRa), 1 μL 1:10-diluted template cDNA, and 0.2 μM of each gene-specific primers, in final Total RNA was extracted from sensitive and tolerant rice volume of 20 μL, using the thermal cycles as follows: 95 °C for materials by Trizol reagent (Invitrogen) after Al3+ treatments, 1 min; 40 or 45 cycles of 95 °C for 10 s; 55 °C for 15 s; and 72 °C and cDNA was reverse transcribed from 1 μg of total RNA for 20 s. Disassociation curve analysis was performed as using M-MLV reverse transcriptase (TaKaRa). Relative quanti- follows: 95 °C for 15 s; 55 °C for 15 s; 94 °C for 1 min. The −ΔΔ fication of gene expression by qPCR was performed on a relative expression levels were calculated by the 2 CT LightCycler® 480 II instrument (Roche). The primers used for method [50]. The reactions were performed in triplicate, and qPCR were designed using Beacon Designer™ 8.0 software the results were averaged. 中国科技论文在线 http://www.paper.edu.cn

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2.7. Determination of sucrose contents 3.2. Potential root protein identification

The rice seedlings were cultured as has been described in To establish the rice roots proteomic changes at the early Section 2.1 of Materials and Methods. Extraction and deter- stage in response to Al toxicity, we employed the robust mination of sucrose contents were conducted as described system of gel-free iTRAQ. Expression profiles of proteins in previously [51], and are described in details in Supplementary roots of both sensitive and tolerant rice plants treated with Materials and Methods. 250 μMAl3+ for 12 h and 24 h were analyzed in two independent 8-plex iTRAQ experiments (for experiment I and experiment II, respectively). The ProteinPilot cut-off score for 3. Results and discussion proteins identified was set at 1.3, which corresponded to a confidence level of 95%. Supplementary Tables S2 and S3 3.1. Evaluation of the Al tolerance for different rice cultivars showed the summary statistics from the search for 12 h and 24 h treatments, respectively. In total, 700 distinct proteins As Al toxicity in acid soils is ever existing during the whole (homologous proteins grouped together) with >95% confi- plant growth season, to evaluate effects of Al3+ on two dence were identified in both experiments (492 proteins from rice cultivars — tolerant cultivar ‘Nipponbare’ and sensitive experiment I and 627 proteins from experiment II, sharing a cultivar ‘7098’, the 4-leaf-old seedlings were pre-grown in a subset of 349 proteins; for detailed protein and peptide 3+ basic solution of 0.5 mM CaCl2 solution and treated with Al information please see Supplementary Tables S4 and S5). concentrations ranging from 0 to 300 μM for 3 days and then Compared to the untreated control plants, 106 (Table 1)of subjected to RRE analysis. Inhibition of root elongation these proteins significantly changed in the sensitive and increased with increasing external Al3+ concentrations in tolerant rice cultivars when they were treated with the toxic 3+ both rice cultivars, and Al tolerance as determined by RRE was Al , within which 62 proteins changed significantly in the always greater in tolerant cultivar than in sensitive cultivar sensitive ‘7098’ and 76 in the tolerant ‘Nipponbare’ plants after 3 day treatments (Fig. 2). Statistically differential effects by sharing 32 proteins (Fig. 3A). The results indicate that 3+ of Al3+ treatments on morphological characteristics of rice different strategies might be adopted in response to Al roots were detected from the concentrations 150 to 300 μM, toxicity in the sensitive and tolerant rice cultivars. with 250 μMAl3+ treatment resulting in the greatest differ- ence between the two cultivars (p < 0.01) (Fig. 2). At the 3.3. Potential proteins regulated by excess Al concentration of 250 μM, the RRE of tolerant cultivar was 49% after 3 day treatment, in contrast to 28% for sensitive cultivar. As the goal of our proteome analysis was to identify proteins 3+ Thus, the Al concentration at 250 μM was used in the in rice root involved in tolerance to excess Al and the following proteomic study. associated tolerance mechanisms, we classified the differentially expressed proteins with reference to the functional categories established by Bevan et al. [47]. Venn diagram and table for the functional classification are shown in Fig. 3B and Table 1, respectively. Broadly, the Al-responsive proteins cover a wide range of molecular functions, including energy (30%), stress and defense (26%), protein turnover (synthesis, modification and degradation) (19%), metabolism (catalyst) (9%), signal transduction (4%), transport and intracellular traffic (4%), cell structure (3%), cell growth/division (2%), transcription (1%), and unknown (2%). The Al-responsive proteins are mainly involved in energy, stress and defense, protein turnover, and metabolism. The results are in accor- dance with the previous reports that the expression of genes involved in energy, stress response and metabolism was up-regulated when exposed to Al3+ stress in aspen [7], Arabidopsis [52], M. truncatula [53], and soybean [54]. In order to obtain a deeper insight into the nature of the proteins that respond to toxic Al3+, we will describe a number of notable differentially regulated proteins listed in Table 1 in detail. Fig. 2 – Effects of Al3+ stress on relative root elongation in tolerant cultivar (Nipponbare) and sensitive cultivar (7098). 3.3.1. Energy related proteins Roots of 4-leaf-old rice seedlings were exposed to 0.5 mM A general symptom of photosynthetic plants under stress is

CaCl2 solution (pH 4.3) containing 0, 50, 100, 150, 200, 250 or energy deficit [55]. Often associated with stress is a reduction μ 300 M AlCl3 for 3 days. The data represents mean ± SD in photosynthesis and/or respiration, which in turn results in (n = 9). Statistically significant differences in different energy deprivation and ultimately in growth arrest and cell sensitive and tolerant cultivars at the same Al3+ treatment death [56]. The energy shortage often leads to the enhance- are indicated with asterisks: (*) p < 0.05 or (**) p < 0.01; ment of inherent pathways of carbohydrate metabolism Student's t-test. and the induction of alternative pathways of glycolysis to 中国科技论文在线 http://www.paper.edu.cn

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Table 1 – List of proteins differentially expressed in sensitive and tolerant rice cultivars treated with Al3+. Accession no. a Protein name % Cov Peptides Ratio d (+Al3+/−Al3+)±SD (95) b (95%) c S (7098) T (Nipponbare)

Energy LOC_Os06g09450 e,f Sucrose synthase 2 51.6 105 1.500 ± 0.085 1.693 ± 0.127 LOC_Os03g28330 f Sucrose synthase 1 42.2 75 0.979 ± 0.059 1.585 ± 0.311 LOC_Os08g02120 e,f Fructokinase-2 51.8 27 1.273 ± 0.082 1.660 ± 0.060 LOC_Os12g25690 e UDP-glucose 6-dehydrogenase 29.4 17 0.678 ± 0.047 0.313 ± 0.188 LOC_Os03g55070 f UDP-glucose 6-dehydrogenase 26.9 13 0.852 ± 0.094 0.492 ± 0.063 LOC_Os05g15770 e Glycosyl hydrolase 23.9 7 0.634 ± 0.229 0.917 ± 0.087 LOC_Os03g53800 f Periplasmic beta-glucosidase precursor 9.3 4 1.058 ± 0.049 0.844 ± 0.043 LOC_Os02g14770 e,f Phosphoenolpyruvate carboxylase 20.8 17 1.621 ± 0.149 1.274 ± 0.094 LOC_Os01g46610 e,f Isocitrate dehydrogenase 25.0 15 1.438 ± 0.332 1.399 ± 0.186 LOC_Os08g09200 f Aconitate hydratase 15.2 15 0.960 ± 0.040 1.321 ± 0.151 LOC_Os02g40830 f Succinyl-CoA ligase beta-chain 8.6 3 0.969 ± 0.040 1.127 ± 0.021 LOC_Os01g49190 f ATP synthase 67.5 79 0.897 ± 0.131 1.482 ± 0.089 LOC_Os04g56160 e,f Plasma membrane ATPase 16.5 17 1.904 ± 0.585 1.972 ± 0.252 LOC_Os06g43850 e,f ATP synthase F1 delta chain 33.3 8 0.798 ± 0.073 1.326 ± 0.067 LOC_Os02g03860 e Mitochondrial ATP synthase precursor 8.3 2 0.887 ± 0.000 0.996 ± 0.014 LOC_Os01g67860 f Fructose-bisphospate aldolase isozyme 53.6 42 0.647 ± 0.086 1.253 ± 0.144 LOC_Os08g03290 f Glyceraldehyde-3-phosphate dehydrogenase 44.5 28 1.046 ± 0.190 2.555 ± 0.200 LOC_Os02g38920 f Glyceraldehyde-3-phosphate dehydrogenase 47.2 40 0.858 ± 0.044 1.213 ± 0.000 LOC_Os02g07260 e Phosphoglycerate kinase 35.6 18 0.655 ± 0.069 0.922 ± 0.051 LOC_Os01g05490 f Triosephosphate isomerase 47.0 13 0.940 ± 0.229 1.315 ± 0.340 LOC_Os11g05110 e,f Pyruvate kinase 17.5 10 1.361 ± 0.181 1.683 ± 0.545 LOC_Os12g05110 f Pyruvate kinase 9.5 4 0.906 ± 0.104 1.203 ± 0.044 LOC_Os06g13810 e,f Pyrophosphate-fructose 6-phosphate 1-phosphotransferase 10.9 10 0.743 ± 0.044 1.453 ± 0.060 LOC_Os03g50480 e,f Phosphoglucomutase 13.8 5 1.213 ± 0.011 0.674 ± 0.182 LOC_Os01g60190 e 2,3-Bisphosphoglycerate-independent 23.4 17 1.405 ± 0.225 0.940 ± 0.285 phosphoglycerate mutase LOC_Os05g40420 e 2,3-Bisphosphoglycerate-independent 18.7 16 0.773 ± 0.021 0.865 ± 0.359 phosphoglycerate mutase LOC_Os02g50620 e Pyruvate dehydrogenase E1 20.0 6 0.875 ± 0.004 0.982 ± 0.036 LOC_Os01g22010 e,f S-adenosylmethionine synthetase 2 50.5 33 0.706 ± 0.039 0.677 ± 0.034 LOC_Os11g26850 e,f Erythronate-4-phosphate dehydrogenase 23.1 11 0.737 ± 0.013 0.813 ± 0.011 LOC_Os03g52840 e Serine hydroxymethyltransferase 10.9 9 1.226 ± 0.330 1.925 ± 0.452 LOC_Os03g60090 f Methylenetetrahydrofolate reductase 14.7 7 0.889 ± 0.049 0.820 ± 0.004 LOC_Os04g53230 f Aminomethyltransferase 7.1 3 0.997 ± 0.060 1.175 ± 0.005

Stress and defense LOC_Os04g59150 e,f Peroxidase precursor 55.8 38 0.632 ± 0.016 1.269 ± 0.296 LOC_Os06g35520 f Peroxidase precursor 47.1 34 1.260 ± 0.213 1.571 ± 0.216 LOC_Os07g01410 e,f Peroxidase precursor 26.3 17 0.647 ± 0.057 1.127 ± 0.010 LOC_Os01g22370 e,f Peroxidase precursor 16.8 10 1.567 ± 0.187 2.063 ± 0.449 LOC_Os05g04380 e Peroxidase precursor 21.4 6 1.562 ± 0.291 1.624 ± 0.465 LOC_Os09g29490 e Peroxidase precursor 7.2 3 0.787 ± 0.007 1.081 ± 0.099 LOC_Os04g59260 e,f Peroxidase precursor 28.4 7 1.275 ± 0.105 0.743 ± 0.044 LOC_Os01g55830 e Glutathione S-transferase 49.3 22 1.538 ± 0.092 1.100 ± 0.033 LOC_Os05g02530 f Glutathione S-transferase 32.7 11 1.108 ± 0.051 1.680 ± 0.208 LOC_Os06g51150 e Catalase isozyme B 22.1 12 0.847 ± 0.000 0.996 ± 0.014 LOC_Os02g02400 e Catalase isozyme A 12.4 6 1.197 ± 0.017 1.410 ± 0.104 e LOC_Os03g17690 L-Ascorbate peroxidase 1 65.2 33 0.586 ± 0.124 0.692 ± 0.315 LOC_Os07g08840 f Thioredoxin H1 27.9 4 0.919 ± 0.188 1.298 ± 0.004 LOC_Os01g70170 e,f Transaldolase 25.2 8 1.255 ± 0.181 1.229 ± 0.102 LOC_Os11g43900 e,f Translationally-controlled tumor protein 14.9 2 0.816 ± 0.071 1.219 ± 0.017 LOC_Os11g06720 e,f Abscisic stress-ripening protein 42.8 15 1.501 ± 0.019 1.992 ± 0.407 LOC_Os01g49290 e,f Guanine nucleotide-binding protein subunit 30.2 15 0.482 ± 0.128 1.209 ± 0.135 beta-like protein LOC_Os08g02080 f Rickettsia 17 kDa surface antigen family protein 26.1 3 0.946 ± 0.009 1.255 ± 0.075 LOC_Os03g18850 e Pathogenesis-related Bet v I family protein 23.1 4 0.758 ± 0.059 0.995 ± 0.186 LOC_Os09g07460 e,f Kelch repeat containing protein 27.1 10 1.292 ± 0.049 1.344 ± 0.227 LOC_Os01g13210 f Salt stress root protein RS1 14.2 3 1.023 ± 0.014 0.759 ± 0.014 LOC_Os02g41590 e Adenosine kinase 34.6 14 0.714 ± 0.117 0.310 ± 0.034 LOC_Os07g09060 e Aldehyde dehydrogenase 17.2 8 1.537 ± 0.169 0.996 ± 0.032 LOC_Os04g58710 e AMP-binding domain containing protein 12.7 3 1.165 ± 0.038 0.986 ± 0.014 LOC_Os03g62620 e,f Late embryogenesis abundant protein 18.0 5 1.171 ± 0.065 1.159 ± 0.021 中国科技论文在线 http://www.paper.edu.cn

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Table 1 (continued) Accession no. a Protein name % Cov Peptides Ratio d (+Al3+/−Al3+)±SD (95) b (95%) c S (7098) T (Nipponbare)

Stress and defense LOC_Os12g42876 f 5-Methyltetrahydropteroyltriglutamate-homocysteine 54.6 112 0.875 ± 0.080 0.867 ± 0.004 methyltransferase LOC_Os10g11500 f Pathogenesis-related protein PRB1 55.7 23 0.830 ± 0.170 0.867 ± 0.004 LOC_Os08g02420 f OsCML7-Calmodulin-related calcium sensor protein 14.9 2 0.881 ± 0.057 0.879 ± 0.008

Protein turnover LOC_Os11g38959 e 40S ribosomal protein S9 26.7 6 0.798 ± 0.081 0.808 ± 0.278 LOC_Os09g31180 e 60S ribosomal protein L6 34.2 6 0.777 ± 0.010 0.851 ± 0.028 LOC_Os04g50990 e,f L11 domain containing ribosomal protein 42.2 6 0.728 ± 0.023 0.650 ± 0.075 LOC_Os09g32976 f 60S ribosomal protein L7 19.0 6 0.968 ± 0.013 0.840 ± 0.023 LOC_Os09g08430 f Ribosomal protein L22 17.0 5 0.918 ± 0.101 0.748 ± 0.099 LOC_Os02g21660 e,f Ribosomal protein 23.6 5 0.817 ± 0.023 0.871 ± 0.008 LOC_Os03g18570 f 40S ribosomal protein S7 30.2 5 0.943 ± 0.048 0.817 ± 0.038 LOC_Os04g51630 f 60S ribosomal protein L7 14.8 3 0.900 ± 0.012 0.863 ± 0.024 LOC_Os07g42450 e 40S ribosomal protein S2 5.6 2 1.149 ± 0.032 0.976 ± 0.081 LOC_Os05g37330 f 60S acidic ribosomal protein 31.0 3 1.171 ± 0.054 1.236 ± 0.011 LOC_Os07g03230 f Eukaryotic translation initiation factor 3 5.8 4 0.986 ± 0.091 0.796 ± 0.044 LOC_Os04g42140 e Eukaryotic initiation factor iso-4 F subunit 2.9 2 0.836 ± 0.027 1.028 ± 0.009 LOC_Os03g08050 f Elongation factor Tu 48.1 51 0.873 ± 0.087 0.661 ± 0.044 LOC_Os02g32030 e,f Elongation factor 20.2 22 1.187 ± 0.299 0.630 ± 0.095 LOC_Os03g11410 e,f Mitochondrial-processing peptidase subunit 19.7 14 0.675 ± 0.105 0.770 ± 0.032 LOC_Os09g39070 e Cysteine protease 13.8 5 0.875 ± 0.004 1.266 ± 0.275 LOC_Os06g50300 f Heat shock protein 12.0 9 0.969 ± 0.137 1.460 ± 0.041 LOC_Os11g09280 e,f OsPDIL1-1 protein disulfide isomerase PDIL1-1 23.8 12 0.709 ± 0.029 1.231 ± 0.199 LOC_Os07g14270 e Calreticulin precursor 20.1 5 0.761 ± 0.063 1.084 ± 0.143 LOC_Os02g22780 f T-complex protein 1 17.4 5 0.872 ± 0.040 1.213 ± 0.011

Metabolism LOC_Os02g41630 f Phenylalanine ammonia-lyase 56.5 57 0.989 ± 0.118 0.631 ± 0.023 LOC_Os03g12290 e,f Glutamine synthetase 38.9 17 1.502 ± 0.083 2.271 ± 0.169 LOC_Os01g55540 f Aspartate aminotransferase 13.7 5 1.049 ± 0.058 1.284 ± 0.059 LOC_Os01g46750 e AMP-binding enzyme 4.7 4 1.112 ± 0.005 0.912 ± 0.017 LOC_Os08g38900 f Tricin synthase 1 29.8 11 1.013 ± 0.057 0.657 ± 0.022 LOC_Os11g32260 f Lysosomal alpha-mannosidase precursor 8.3 5 1.172 ± 0.075 1.208 ± 0.017 LOC_Os01g42880 e Taxadienol acetyl transferase-like 11.6 5 1.368 ± 0.132 0.848 ± 0.199 LOC_Os01g32080 e Thiamine pyrophosphate enzyme 10.8 4 1.193 ± 0.055 1.184 ± 0.107 LOC_Os01g24680 f 3-Hydroxyacyl-CoA dehydrogenase 8.0 4 0.951 ± 0.013 0.813 ± 0.019 LOC_Os01g40860 f Aldehyde dehydrogenase 8.4 5 0.989 ± 0.068 1.170 ± 0.011

Signal transduction LOC_Os04g38870 e 14-3-3 protein 44.7 23 1.303 ± 0.078 1.220 ± 0.265 LOC_Os03g13540 f Purple acid phosphatase 1 11.5 3 1.033 ± 0.032 1.197 ± 0.038 LOC_Os03g12250 e,f Atypical receptor-like kinase MARK 7.1 4 0.802 ± 0.018 0.570 ± 0.016 LOC_Os08g09940 e GTP-binding protein 11.4 3 0.802 ± 0.035 1.102 ± 0.005

Transporters and intracellular traffic LOC_Os07g26690 e Aquaporin PIP2 20.0 2 1.171 ± 0.065 0.398 ± 0.117 LOC_Os02g51110 e,f Aquaporin NIP2 7.4 2 1.310 ± 0.204 1.357 ± 0.192 LOC_Os05g45950 e,f Mitochondrial outer membrane protein porin 12.1 2 0.836 ± 0.035 1.405 ± 0.123 LOC_Os10g40530 f Cortical cell delineating protein 12.1 2 1.062 ± 0.005 0.875 ± 0.004

Cell structure LOC_Os05g38640 f Histone H2A 35.6 9 1.282 ± 0.378 0.677 ± 0.162 LOC_Os10g39410 e,f Histone H4 51.5 8 1.313 ± 0.042 1.312 ± 0.124 LOC_Os11g03730 f Alpha-N-arabinofuranosidase A 12.8 10 1.049 ± 0.120 1.546 ± 0.377

Cell growth/division LOC_Os03g05730 f Cell division control protein 48 homolog E 16.2 11 1.312 ± 0.216 1.152 ± 0.281 LOC_Os02g06290 f Senescence-associated protein-like protein 17.3 2 0.918 ± 0.139 2.093 ± 0.306

Transcription LOC_Os06g51220 f HMG1/2 protein 42.7 5 1.041 ± 0.289 1.952 ± 0.099

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Table 1 (continued) Accession no. a Protein name % Cov Peptides Ratio d (+Al3+/−Al3+)±SD (95) b (95%) c S (7098) T (Nipponbare)

Unclassified LOC_Os07g45080 e,f Expressed protein 21.0 3 0.756 ± 0.031 1.743 ± 0.271 LOC_Os07g07990 f Uncharacterized protein 7.7 2 0.927 ± 0.110 1.291 ± 0.077

a Accession no. is the locus name of a gene in the RGAP (Rice Genome Annotation Project Database) release 7. b % Cov (95) indicates the percentage of matching amino acids from identified peptides having confidence greater than or equal to 95%. c Peptides (95%) indicate the number of distinct peptides having at least 95% confidence. d The values were calculated as the ratio of 114, 118 (Al3+-treated sensitive cultivar) to 113, 117 (sensitive cultivar) label; 116, 121 (Al3+-treated tolerant cultivar) to 115, 119 (tolerant cultivar) label. Significant changes are indicated by boldface. e Proteins were observed in the sensitive cultivar. f Proteins were observed in the tolerant cultivar.

maintain energy and carbon skeletons for key metabolic metabolism were identified (Table 1), providing evidence processes. Sucrose is the primary translocatable carbohydrate that regulation of sucrose metabolism and homeostasis is in the majority of plants, thus, its metabolism is vital to involved in plant response to Al3+ stress. The expression of the the regulation of plant growth and response to stresses [57]. sucrose synthase 1 in tolerant cultivar was higher than in In this proteomic study, seven proteins, including two sensitive cultivar, while the expression of both two UDP-glucose sucrose synthases (LOC_Os06g09450 and LOC_Os03g28330), 6-dehydrogenases was opposite in tolerant and sensitive two UDP-glucose 6-dehydrogenases (LOC_Os12g25690 and cultivars, indicating that the tolerant cultivar could maintain LOC_Os03g55070), a fructokinase (LOC_Os08g02120), glycosyl sucrose at a higher level through sucrose synthases by com- hydrolase (LOC_Os05g15770), and periplasmic beta-glucosidase peting their common substrate, UDP-glucose, with UDP-glucose precursor (LOC_Os03g53800), which are related to sucrose 6-dehydrogenases. In the stop1 mutant of Arabidopsis,the disturbance of sucrose metabolism would result in more sensitive to Al [58]. Therefore, we propose that sucrose might act as an important mediator in plant response to Al3+ stress, and the elevation of sucrose metabolic level may confer the plant tolerance to Al3+ toxicity. To confirm this hypothesis, we measured the contents of sucrose in roots of both tolerant and sensitive cultivars. Results showed that the sucrose contents increased in a time-dependent manner in both tolerant and sensitive rice cultivars under the stress of Al toxicity (Fig. 4). Interestingly, under control culture conditions (without Al), the sucrose content in sensitive cultivar was only approximately two-thirds as much as that in the tolerant cultivar, which was responsible for sensitivity of sensitive cultivar to toxic Al. These results strongly indicated that sucrose concentration or its metabolism plays an important role in Al tolerance in plants. The tricarboxylic acid (TCA) cycle is one of the iconic pathways in metabolism and it is commonly thought of in terms of energy metabolism, being responsible for the oxidation of respiratory substrates to drive ATP synthesis [59]. It has been demonstrated that modification of TCA cycles is of crucial significance in plant adaptation to unfavorable environments [60].Inthe present study, three identified proteins, isocitrate dehydrogenase (LOC_Os01g46610), aconitate hydratase (LOC_Os08g09200), and succinyl-CoA ligase beta-chain (LOC_Os02g40830) (Table 1)with Fig. 3 – Venn diagram analyses of the differentially different expression patterns in sensitive and tolerant cultivars, expressed proteins. (A) Venn diagram analysis of the are involved in this cycle, suggesting that an alternative TCA differentially expressed proteins identified in rice roots. The cycle may also play a crucial role in the adaptation of the rice numbers of the differentially expressed proteins identified in plants to Al3+ stress. In addition, four ATP synthases (ATPase) sensitive and tolerant varieties are shown in the different (LOC_Os01g49190, LOC_Os04g56160, LOC_Os06g43850 and segments. (B) Classification of the differentially expressed LOC_Os02g03860) (Table 1) were also identified. Plant ATPases proteins into molecular functions. The pie chart shows the located to different subcellular organelles have different distribution of the differentially expressed proteins into their functions, including proton transporting, sucrose translocation, functional classes in value and percentage. stomata opening, plant growth, intracellular pH homeostasis, 中国科技论文在线 http://www.paper.edu.cn

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providing evidence that a complete glycolytic pathway [63] is involved in response to Al3+ stress. Glycolysis is a catabolic anaerobic pathway in which oxidizes hexoses to generate ATP, reductant, and pyruvate, and also produces building blocks for anabolism [64]. It is the predominant pathway that “fuels” plant respiration. In this study, most of the identified glycolysis related proteins were up-regulated in the tolerant cultivar, indicating that the tolerant cultivar could maintain their essential respiration and provide more glycolytically- generated ATP by strengthening the glycolytic pathway under Al3+ stress. In mammalian astrocytes, glycolytically-generated ATP is used to increase their mitochondrial membrane potential to resist pro-apoptotic stimuli and thus made them survival under stress [65,66]. Therefore, it is reasonable to assume that glycolytically-generated ATP may also play an important role in protecting plants from Al3+ toxicity. Taken all together, the tolerant rice plants appear to be able to perceive and convert stress signaling into energy status in a skillful manner and induce alternative metabolic pathways and thus adjust their growth and development in response to Al3+ stress.

3.3.2. Stress and defense related proteins Plants have developed defense responses to abiotic and biotic stresses over evolutionary time as a survival mechanism [67].A set of 28 proteins, accounting for 26% of all differentially regulated proteins, belong to this category, and fourteen of them are involved in redox homeostasis. Generation of reactive oxygen species (ROS) is one of the first universal reactions to abiotic or biotic challenges in plants [68,69]. Proteins and metabolites with redox and antioxidant properties are therefore essential not only Fig. 4 – Sucrose contents of roots in tolerant cultivar for the maintenance of functional roots during their long lifetime (Nipponbare) and sensitive cultivar (7098). Roots of 4-leaf-old but also for manipulation of Al3+ toxicity [70,71]. Seven peroxidase rice seedlings were exposed to 0.5 mM CaCl2 solution proteins (LOC_Os04g59150, LOC_Os06g35520, LOC_Os07g01410, μ (pH 4.3) containing 150 and 250 M AlCl3 for 0, 12, 24 and LOC_Os01g22370, LOC_Os05g04380, LOC_Os09g29490 and LOC_ 48 h. Data are given as means ± standard deviation (SD) of Os04g59260) (Table 1) with putative function of producing ROS three biological replicates. Significant differences of sucrose were detected in our iTRAQ data, which were previously contents between 12, 24, 48 h and 0 h after the Al treatments identified in the roots of Arabidopsis [52],wheat[63],andaspen are indicated by **p < 0.01; Student's t-test. [7] under Al3+ stress. Antioxidant proteins were also identified, for example, two glutathione S-transferases (GSTs) (LOC_Os01g55830 and LOC_Os05g02530), two catalase isozymes (CATs) (LOC_ redox regulation and stress response [61]. The four identified Os06g51150 and LOC_Os02g02400), a thioredoxin (LOC_ ATPases were all expressed at a higher level in tolerant cultivar Os07g08840), L-ascorbate peroxidase (APX) (LOC_Os03g17690) than that in sensitive cultivar during Al3+ stress. This observa- and transaldolase (LOC_Os01g70170) (Table 1), and some of tion is in line with previous report that vacuolar ATPase and which were firstly detected. Therefore, both oxidant and antiox- mitochondrial ATPase were induced by Al in an Al-resistant idant proteins appeared to be present in the rice root proteome wheat cultivar [62]. It is highly likely that the tolerant cultivar when they were exposed to toxic Al3+. can generally maintain ATPases at a higher level, thus facilitate In addition to the redox related proteins, a translationally- the plant to operate related biological processes to counteract controlled tumor protein (TCTP) (LOC_Os11g43900), abscisic Al3+ toxicity. stress-ripening protein (LOC_Os11g06720), guanine nucleotide- Furthermore, in the present proteomic study, a binding protein subunit beta-like protein (LOC_Os01g49290), fructose-bisphospate aldolase isozyme (LOC_Os01g67860), two rickettsia 17 kDa surface antigen family protein (LOC_ glyceraldehyde-3-phosphate dehydrogenases (LOC_Os08g03290 Os08g02080), and pathogenesis-related Bet v I family protein and LOC_Os02g38920), phosphoglycerate kinase (LOC_ (LOC_Os03g18850) were expressed at higher level in the tolerant Os02g07260), triosephosphate isomerase (LOC_Os01g05490), cultivar than in the sensitive cultivar (Table 1). Compared to the two pyruvate kinases (LOC_Os11g05110 and LOC_Os12g05110), above mentioned proteins, a salt stress root protein RS1 pyrophosphate-fructose 6-phosphate 1-phosphotransferase (LOC_Os01g13210), adenosine kinase (LOC_Os02g41590), alde- (LOC_Os06g13810), phosphogucomutase (LOC_Os03g50480), hyde dehydrogenase (LOC_Os07g09060), AMP-binding domain two 2,3-bisphosphoglycerate-independent phosphoglycerate containing protein (LOC_Os04g58710), and Late embryogenesis mutases (LOC_Os01g60190 and LOC_Os05g40420) and pyruvate abundant protein (LOC_Os03g62620) were expressed at higher dehydrogenase E1 (LOC_Os02g50620) (Table 1) were identified, level in the sensitive cultivar than in the tolerant cultivar 中国科技论文在线 http://www.paper.edu.cn

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(Table 1). Interestingly, most of them play diverse roles in and drought stress tolerance compared to those of control [79]. various stress responses. For example, TCTP, up-regulated in In addition, several proteins that facilitate the proper folding of resistant lines in our experiment, has previously been identified newly synthesized proteins or refolding of the damaged in soybean exposed to Al3+ stress in a proteomic study [67], proteins were detected in this proteomic study. A heat shock which is an important component of TOR (target of rapamycin) protein (LOC_Os06g50300), protein disulphide isomerase-like signaling pathway and regulates plant growth and stress protein 1 (PDIL1) (LOC_Os11g09280), calreticulin precursor response [72] The pathogenesis-related protein has also been (LOC_Os07g14270) and T-complex protein 1 (LOC_Os02g22780) proven to participate in Al3+ tolerance at the protein level, and whose expression levels in the tolerant was higher than in the whose transcriptional level was elevated under Al3+ stress [67]. sensitive cultivar after Al3+ treatment (Table 1). Altogether, when exposed to toxic Al3+, the tolerant cultivar could 3.3.3. Protein homeostasis related proteins reprogram the protein translation and replenish the respiratory Protein turnover, the balance between synthesis and degrada- pathway to make them survive better under stress condition tion, is one of many forms of regulation that are coordinated to compared to the sensitive cultivar. achieve a unified cellular response to developmental and environmental cues [73]. Twenty proteins implicated in protein 3.3.4. Proteins related to metabolism translation, processing, and degradation were identified in the Metabolic proteins related to amino acid metabolism, nitrogen present study (Table 1). Previous study has shown that assimilation, nucleotide anabolism, and lipid oxidation were translation is one of the main targets of growth control in also identified in the present proteomic study. For example, a response to changes in the environment [74]. In our iTRAQ data, phenylalanine ammonia-lyase (PAL) (LOC_Os02g41630), which eight ribosomal proteins were down-regulated in both tolerant catalyzes the nonoxidative deamination of L-phenylalanine and sensitive cultivars while two ribosomal components were to form trans-cinnamic acid and a free ammonium ion and up-regulated in the tolerant or sensitive cultivars (Table 1). This channels the carbon from primary metabolism into phenyl- was consistent with previous microarray data showing that propanoid secondary metabolism in plants [80], was identified transcript abundances of the majority of ribosomal proteins in the present study (Table 1). To our knowledge, it was first to were degressive and transcripts for specific ribosomal compo- be identified in response to Al3+ stress at the protein level. It was nents were enhancive during Al3+ exposure in Arabidopsis [52]. consistent with previous report that a rapid loss of PAL occurred In addition, two translation initiation factors (LOC_Os07g03230 in bean cell suspension cultures when treated with the elicitor and LOC_Os04g42140) and two elongation factors (LOC_ [81]. Furthermore, it is interesting to note that a glutamine Os03g08050 and LOC_Os02g32030) were also identified in our synthase (GS) (LOC_Os03g12290), which catalyzes the conver- study (Table 1). Interestingly, the expression of a eukaryotic sion of ammonia and glutamate to glutamine, was up-regulated translation initiation factor 3 (eIF3) (LOC_Os07g03230) was in both sensitive and tolerant cultivars (Table 1). It is a key down-regulated in the tolerant cultivar while nearly main- enzyme of primary N assimilation, as well as ammonia tained the same level in the sensitive cultivar, suggesting that reassimilation [82] and detoxification of metal stress [83].It the translation was reasonably controlled in the tolerant also has been shown that plant GS promotes organic acid and cultivar to save the energy. This was in line with a previous Al3+ to bound to the polypeptide structure of GS molecule to report that the expression of eIF3 was down-regulated in detoxify cytoplasmic Al3+ in wheat [84]. This observation Arabidopsis when exposed to salt stress [75].Furthermore,two suggests that the GS protein may play an important role in elongation factors were also expressed at a lower level in the response to Al3+ toxicity and confers resistance in both sensitive tolerant than in sensitive cultivar. The differential regulation of and tolerant cultivars. different components of the translation machinery suggested that there was a complicated mechanism controlling protein 3.3.5. Signal transduction and transporters related proteins synthesis in response to Al3+ stress. Compared to the sensitive Perception and transmission of stress signals are the central cultivar, the protein synthesis was tightly controlled in the piece of plant response to environmental stresses. Several tolerant cultivar which resulted in conserving more resources components of signaling pathways identified in our iTRAQ data that were needed to survive under Al3+ stress. were responsive to Al3+ toxicity, including putative sensors, Protein degradation is a routine when plants are continu- protein phosphatases, as well as protein kinases, for example, ously confronted with unfavorable environmental conditions. 14-3-3 protein (LOC_Os04g38870) and purple acid phosphatase 1 This response cannot only provide molecular substrates for (PAP1) (LOC_Os03g13540) (Table 1). It has been shown that plant respiration but also initiate adaptive responses to abiotic 14-3-3 proteins are implicated in stress responses in rice [85]. and biotic stresses [76]. Here, two proteins (LOC_Os09g39070 Here, we identified a 14-3-3 protein (LOC_Os04g38870) which and LOC_Os03g11410) involved in protein proteolysis were was up-regulated in both the sensitive and tolerant cultivars. identified in the present study (Table 1). It is interesting to This suggested that it may be involved in signal transmitting note that a cysteine protease (LOC_Os09g39070) was expressed in response to Al3+ stress. Previous study has demonstrated at a higher level in the tolerant than in the sensitive cultivar, that Al toxicity usually causes plant phosphorus (P) deficiency suggesting that it may play a vital role in resisting against toxic stress on acid soils [2] and thus induces purple acid phospha- Al3+. This observation was consistent with previous studies tase (PAP) to enhance P acquisition and utilization in higher which showed that plant cysteine proteases were important plants [86]. In our study, PAP1 expressed at a higher level in the players in plant resistant response to pathogen infection and tolerant than in the sensitive cultivar, suggesting that the insect attack [77,78], and transgenic Arabidopsis plants with tolerant cultivar can relieve the Al3+ toxicity through improving constitutive cysteine protease expression showed higher salt the P absorption. 中国科技论文在线 http://www.paper.edu.cn

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Regulated influx and efflux of solutes and various macro- Previous work has shown that pathogen infection activates molecules across biological membranes is an essential genes that are normally induced during senescence, suggest- component of cellular stress responses. Two aquaporin ing that senescence may be involved with plant defense [93]. proteins (LOC_Os07g26690 and LOC_Os02g51110), mitochon- Here, we also identified a senescence-associated protein-like drial outer membrane porin 2 (LOC_Os05g45950) and lipid protein (SALP) (LOC_Os02g06290) (Table 1) whose expression transfer protein (LTP) (LOC_Os10g40530) were identified in level in the tolerant was twice more than that in the sensitive, this proteomic study (Table 1). Aquaporins are channel suggesting that Al3+ stress may induce leaf senescence in proteins that facilitate the transport of water and small tolerant cultivar. This was consistent with previous observa- neutral molecules, including gases, across cell membranes of tion that a list of senescence-associated genes would be most of the living organisms and they play a vital role in induced under salt stress [94]. Possibly, this response could maintaining the water and nutrient status of the plants [87].It help the plant adapt to Al3+ stress in a number of ways, has been shown that plant aquaporin PIP2 is a high-efficiency including decreased transpiration in senescent leaves, diver- water channel responsible for cellular water transport in roots sion of energy and food resources from growth to defense and [88]. In our study, the expression of aquaporin PIP2 was protection of important organs by accumulation of toxic ions down-regulated by approximately 50% in the tolerant cultivar, in the senescent leaves. while it was scarcely changed in the sensitive cultivar. This In plants, it has been shown that chromatin mediated suggested that the tolerant was able to maintain the water regulation of gene expression plays an important role in the potential for basic physiological and biochemical reactions response to abiotic stresses, and it appears that histone through down-regulation of aquaporin PIP2 in response to proteins and high mobility group (HMG) proteins are involved Al3+ stress. These observations suggested that the regulation in the stress-induced reactions [95]. Here two histone proteins of water by aquaporins may be an important early response to (LOC_Os05g38640 and LOC_Os10g39410) and a high mobility Al3+ stress. Mitochondrial porin, which is also known as the group B protein (HMGB) (LOC_Os06g51220) were identified voltage dependent anion-selective channel (VDAC), is the (Table 1). Histone H4 was up-regulated at the similar level in most abundant protein in the mitochondrial outer membrane both sensitive and tolerant cultivars, while histone H2A and the major transport pathway for a large variety of showed contrasting patterns of up- and down-regulated compounds ranging from ions to large polymeric molecules expression. This suggests that the strategy of nucleosome such as DNA and tRNA [89]. Previous study has shown that assembly in the tolerant cultivar differs from that in the rice VDAC genes are up-regulated in roots during the recovery sensitive one, and is consistent with previous observations in period after stress release [90]. Here, we also identified a M. truncatula [53] and soybean [67]. It has been reported that mitochondrial outer membrane porin 2 (LOC_Os05g45950) the HMGB proteins are involved as architectural factors in whose expression in the tolerant cultivar was higher than various DNA-dependent nuclear processes, including tran- that in the sensitive cultivar, indicating that the tolerant scription, recombination, and DNA repair [96]. In our iTRAQ cultivar could rapidly recover from Al3+ toxicity. data, the expression of HMGB protein in the tolerant was nearly twice higher than that in the sensitive, indicating that 3.3.6. Proteins related to cell structure, cell growth/division and HMGB proteins may play an essential role in response to Al3+ transcription stress in the tolerant rice cultivar. Cell walls undergo dynamic changes during plant growth and development or in response to biotic and abiotic stress 3.4. Identification biochemical reactions significantly [91].Anα-L-arabinofuranosidase (LOC_Os11g03730) involved up-regulated by excess Al in this process was identified in this study (Table 1). α-L-Arabinofuranosidases are capable of releasing terminal To gain insight into up-regulated metabolic pathways in rice 3+ nonreducing α-L-arabinofuranosyl residues from cell wall roots during Al stress, we conducted the KOBAS analysis. matrix polymers, as well as from different glycoconjugates Fifty-two significantly up-regulated proteins (ratio > 1.2 in [92]. α-L-Arabinofuranosidase was expressed at a higher level in sensitive cultivar or tolerant cultivar, or ratio > 1.1 in both the tolerant cultivar than in the sensitive cultivar, indicating sensitive cultivar and tolerant cultivar) (Supplementary Table that it may play an essential role in resisting Al3+ through the S6) identified in our iTRAQ data, having E values higher than modification of cell walls. Furthermore, some proteins identi- the cutoff in the KEGG pathway database, were subjected to fied related to sucrose metabolism enzymes were also involved KOBAS analysis and adjusted manually, and KOBAS 2.0 in cell wall modification, for example, the glycosyl hydrolase mapped them into 35 KEGG pathways. Four of these pathways (LOC_Os05g15770) and beta-glucosidase (LOC_Os03g53800) were significantly up-regulated (p < 0.05) in response to Al3+ (Table 1). It has been shown that sucrose-hydrolyzed UDP- stress if judged by p-value based on hypergeometric distribu- glucose partakes in cell wall modification and plays an tion. Two of these pathways had p-values <0.05 after FDR important role in Al tolerance in rice [26]. Exogenous UDP- correction (Table 2). Glycolysis/Gluconeogenesis, which is an glucose could significantly alleviate the Al-induced inhibition of alternative bioenergetic pathway in living organisms under root growth in the star1 mutant which was sensitive to toxic Al stress conditions, was ranked number one (Table 2). A [26], which, together with our results, indicated that sucrose detailed view of the glycolytic/gluconeogenetic pathway was metabolism and homeostasis are also involved in plant shown in Fig. 5. Eight proteins, LOC_Os03g50480 (EC: 5.4.2.2), response to Al3+ stress by remodeling the cell wall. LOC_Os06g13810 (EC: 2.7.1.90), LOC_Os01g05490 (EC: 5.3.1.1), When plants are exposed to environmental stresses, they LOC_Os08g03290 (EC: 1.2.1.12), LOC_Os02g38920 (EC: 1.2.1.12), often show senescent phenotypes such as leaf chlorosis. LOC_Os01g60190 (EC: 5.4.2.1), LOC_Os11g05110 (EC: 2.7.1.40), 中国科技论文在线 http://www.paper.edu.cn

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Table 2 – Representative Al-preferential metabolic pathways identified by KOBAS. Serial KEGG pathway(s) a KEGG ID No. of rice proteins located No. of up-regulated p-Value b FDR-corrected no. in various pathways proteins in rice cultivars p-value

Total – 3940 52 –– 1 Glycolysis/gluconeogenesis osa00010 115 8 0.0004 0.0128 2 Carbon fixation in photosynthetic osa00710 79 6 0.0013 0.0226 organisms 3 Fructose and mannose metabolism osa00051 53 4 0.0087 0.1019 4 Pentose phosphate pathway osa00030 45 3 0.0316 0.2766

a All KEGG pathways were retrieved from KEGG release 61.1 on February 1, 2012. b Pathways with a p-value higher than 0.05 were not listed.

Fig. 5 – Regulatory changes in the pathway of glycolysis/gluconeogenesis. Colored rectangles correspond with the rice genes detected in the iTRAQ data. Green, down-regulated; yellow, up-regulated. 中国科技论文在线 http://www.paper.edu.cn

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and LOC_Os12g05110 (EC: 2.7.1.40) were involved in the steps astrocytes, glycolytically-generated ATP is used to increase of 1, 3, 5, 6, 8 and 10 in glycolysis/gluconeogenesis and colored their mitochondrial membrane potential to resist pro-apoptotic by yellow. These steps are considered to be regulated or stimuli and thus made them survive under stress [65,66]. rate-limiting steps and, their up-regulated expressions may Collectively, our proteomic data and KOBAS analysis provide play a vital role in starting the glycolytic/gluconeogenetic the evidence that glycolytically-generated ATP also plays an pathway effectively towards unfavorable environments. This important role in protecting plants from Al3+ toxicity. was consistent with previous observation that a list of glycolysis/ gluconeogenesis related genes would be induced at transcrip- 3.5. Verification of iTRAQ data on selected candidates by qPCR tional level under Al stress in wheat [62]. As we discussed earlier, the promotion of the glycolytic/gluconeogenetic pathway could To obtain complementary information related to the above result in more glycolytically-generated ATP. In mammalian iTRAQ data and KOBAS results, we also examined expression

Fig. 6 – qPCR data for the mRNA expression levels of genes for Al-responsive proteins mapped in glycolytic/gluconeogenetic pathway in roots of tolerant and sensitive rice cultivars. The values represented relative mRNA levels against control groups (0 h samples), values of which were all set to 1 unit. Statistically significant differences in gene expression are indicated with asterisks: (*) p < 0.05 or (**) p < 0.01; Student's t-test. 中国科技论文在线 http://www.paper.edu.cn

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levels of these genes involved in glycolysis/gluconeogenesis. RRE relative root elongation RNA of the sensitive and tolerant cultivars was extracted FDR false discovery rate from 0 to 24 h of Al3+ treatments, followed by qPCR analysis. PSPEP proteomics system performance evaluation pipeline The expression patterns of the eight genes – LOC_Os03g50480, EF error factor LOC_Os06g13810, LOC_Os01g05490, LOC_Os08g03290, LOC_ KEGG kyoto encyclopedia of genes and genomes Os02g38920, LOC_Os01g60190, LOC_Os11g05110, and LOC_ KOBAS KEGG orthology-based annotation system. Os12g05110 – were summarized in Fig. 6. qPCR results showed that upon Al3+ stress, the eight genes encoding key enzymes related to glycolysis/gluconeogenesis had significant up- regulation in the case of both sensitive and tolerant rice Conflict of interest plants (Fig. 6), which was in line with the iTRAQ data; such remarkable activation of glycolytic/gluconeogenetic pathway The authors declare that they have no conflict of interest. suggests strong promotion of biosysnthesis of available energy since the glycolysis/gluconeogenesis is the predominant pathway fueling plant respiration. Interestingly, as early Acknowledgments as 3 h of Al3+ treatments, the eight genes were induced and had a significant higher expression level in tolerant cultivar The authors gratefully acknowledge Prof. Jian Feng Ma in compared with sensitive cultivar, whereas significant up- Okayama University (Kurashiki, Japan) for gifting the seeds of 3+ regulation was observed until after 6 h Al treatment in ‘7098’. This work was supported by the National Natural sensitive cultivar (Fig. 6). These results suggest that the Science Foundation of China (Grant No. 31210103907), the tolerant cultivar was capable of perceiving environmental Changjiang Scholarship and Innovative Research Team (Grant stress and enhancing energy supply earlier than the sensitive No. IRT1185) and the Fundamental Research Funds for the 3+ cultivar under the Al stress. For tolerant cultivar, it is timely Central Universities. stress response that makes it more survivable under toxic Al3+ condition compared to the sensitive cultivar. REFERENCES

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