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Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558
Journal of Integrative Agriculture
March 2013
2013, 12(3): 398-405
RESEARCH ARTICLE
Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558 ZHENG Yong-sheng1, MA Xiao-gang2, CHI De-zhao2, GAO Ai-nong1, LI Li-hui1 and LIU Wei-hua1 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2 Qinghai Academy of Agriculture and Forestry, Xining 810000, P.R.China 1
Abstract Spike number is one of three yield-related factors and is closely related to wheat yield. In the present study, we found that the inhibited and normal tillers of the 3558 line presented phenotypic differences at the elongation stage by morphological and anatomical analysis. We then initiated a proteomic study using two-dimensional electrophoresis (2-DE) and nanoscale liquid chromatography-high-definition tandem mass spectroscopy, to isolate and identify the key proteins and metabolic pathways related to spike-development inhibition. A total of 31 differentially expressed proteins (DEPs), which were mainly involved in cell cycle regulation, photosynthesis, glycolysis, stress response, and oxidation-reduction reactions, were isolated and identified. 14-3-3-like proteins and proliferating cell nuclear antigen (PCNA), involved in cell-cycle regulation, were dramatically down-regulated in inhibited tillers compared to normal tillers. Six spots corresponding to degraded Rubisco large subunits, involved in photosynthesis, were detected in different locations of the 2-DE gels and were up-regulated in inhibited tillers. In addition, the relative levels of DEPs involved in glycolysis and oxidationreduction reactions changed dramatically. Development was blocked or delayed at the elongation stage in the inhibited tillers of 3558. Weakened energy metabolism might be one reason that the inhibited tillers could not joint and develop into spikes. These DEPs and related metabolic pathways are significant for understanding the mechanism of spike-development inhibition and studying the spike-development process in wheat. Key words: wheat, spike-development inhibition, 2-DE, differentially expressed proteins
INTRODUCTION The problem of food security has gained attention because of the continued growth of the population, substantial reduction of agricultural land, climate change, and frequent pest and disease occurrences. Wheat is one of the most important food crops and plays an irreplaceable role in improving and stabilizing food production to address food security issues. In recent years, because wheat acreage has declined and production is
at standstill, the wheat supply and demand relationship had become increasingly acute. Improving wheat yield through genetic alteration has become one of the most prominent areas of study for wheat breeders and scientists. Panicles (spikes) per unit area, number of spikelets per panicle and grain weight are three important yield components for crop plants. The number of spikes per unit area has been found to be the most important component of yield, accounting for 87% of the variation in yield (Gravois and McNew 1993). Highyield breeding should therefore focus on increasing the
Received 2 May, 2012 Accepted 24 September, 2012 ZHENG Yong-sheng, Tel: +86-531-83178713, E-mail: [email protected]; Correspondance LIU Wei-hua, Tel: +86-10-62176077, Fax: +86-10-62189650, E-mail: [email protected] © 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60239-7
Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558
number of spikes per unit area. Tillering in cereal crops such as rice and wheat involves two stages: the formation of axillary buds and the subsequent outgrowth of the buds (Hanada 1993). The formation of axillary buds determines the tillering capacity and is potentially controlled by a single gene, while the development of the buds into spikes is a complex developmental process (Li et al. 2003). In bread wheat, the genetic mechanisms of tillering capacity have been explored. Richards (1988) reported a low-tillering wheat mutation called Israel uniculm-492, which was controlled by one recessive gene, tin; tiller formation was also blocked or affected by high temperature, day length, and extreme cultivation conditions (Richards 1988). The recessive gene has been mapped to the short arm of chromosome 1A (Spielmeyer and Richards 2004). A dominant gene, tin2, which controls the oligoculm phenotype, was located on chromosome 2A as determined by the genetic analysis of the Chinese Spring (CS) monosomic series (Peng et al. 2007). Kuraparthy et al. (2007) isolated a wheat mutant that produced one main culm and lost tillering capability using ethyl methanesulphonate (EMS)-based mutagenesis in diploid wheat. The mutant single recessive gene tin3 was located on the long arm of chromosome 3A and was found to be closely linked with Xpsr1205 (Kuraparthy et al. 2007). Although the wheat genes influencing the formation of axillary buds have been described, the mechanism of tiller development into spikes in wheat is largely uncharacterized. The wheat spike-inhibition phenotype of 3558 is controlled by a single recessive gene which was termed the fertile tiller inhibition gene and was mapped to chromo-
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some 1AS (Zhang et al. 2012). In the present study, the wheat mutant line 3558, which has normal tillering capacity (Fig. 1-A) but a spike-development inhibition phenotype (Fig. 1-C), was used to analyze the key proteins and metabolic pathways correlated with spike development using two-dimensional electrophoresis (2-DE) and nano-scale liquid chromatography-high-definition tandem mass spectroscopy. Our study will increase our understanding of spike development in wheat and provide a genetic basis to improve wheat yield.
RESULTS 2-DE mapping, protein spot identification and functional classification To delineate the key DEPs and metabolic pathways underlying the phenotypic differences between normal and inhibited tillers of 3558 line, we analyzed their proteomes at the elongation stage (Fig. 1-B). A total of (1 500±50) spots were reproducibly detected in the three gel replicates (Fig. 2). For the inhibited tillers, 35 spots had significantly altered intensities compared with the corresponding normal tillers (P<0.05), and the intensities for these spots increased by more than 1.5 fold. For the inhibited tillers, 13 spots were significantly upregulated and 22 were down-regulated. A total of 35 differentially expressed protein (DEPs) spots were analyzed by NanoLC-HDMS MS/MS, and 31 spots were successfully identified (Table). The 31 DEPs were grouped into 6 categories according to their functional annotations in the UniProtKB
Fig. 1 Morphology and anatomy of line 3558. A, seedling morphology of line 3558 at the elongation stage. Arrows indicate an inhibited tiller (1) and a normal tiller (2). B, the anatomies of inhibited (left) and normal (right) tillers. C, the morphologies of line 3558.
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Fig. 2 2-DE reference maps of normal and inhibited tillers at the elongation stage. A, 2-DE map of the normal tiller proteome. B, 2-DE map of the inhibited tiller proteome. C, key DEP spots.
database. They were mainly involved in cell cycle regulation (11 spots), photosynthesis (9 spots), glycolysis (4 spots), stress response (3 spots), and oxidation-reduction reactions (3 spots, Table 1).
DEPs involved in cell cycle regulation were all down-regulated Eleven of the identified DEPs were involved in cell cycle regulation. They were all down-regulated in the spikedevelopment inhibition tillers. Among these DEPs, six were alpha and beta subunits of tubulin: two isoforms of the tubulin beta-5 subunit (spots 50 and 56), two
isoforms of the alpha tubulin-4D subunit (spots 58 and 59), one tubulin beta subunit (spot 52), and one tubulin alpha subunit (spot 57). One additional spot, identified as actin-2, was also down-regulated. Tubulin and actin form the microtubules and microfilament, respectively, and constitute the cell cytoskeleton. They have important roles in maintaining the cell’s form and the process of cell division. Proliferating cell nuclear antigen (PCNA, spot 92) was drastically down-regulated in the inhibited tillers. This protein is a DNA polymerase accessory factor required for DNA replication, and its expression is an indicator of cell proliferation (Gary et al. 1997). The
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Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558
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Table Differentially expressed proteins in the normal and inhibited tillers of the 3558 line at the elongation stage Spot no. 1)
Protein name
Cell structure and cell cycle regulation 50 Tubulin beta-5 chain 52 Beta-tubulin 56 Tubulin beta-5 chain 57 Alpha tubulin 58 Alpha tubulin-4D 59 Alpha tubulin-4D 23 Actin-2 53 Proliferating cell nuclear antigen 30 14-3-3-like protein B 31 14-3-3 protein 32 14-3-3 protein homologue Photosynthesis 1 RuBisCO large subunit 11 RuBisCO large subunit 16 RuBisCO large subunit 33 RuBisCO large subunit 37 RuBisCO large subunit 40 RuBisCO large subunit 20 RuBisCO large subunit-binding protein subunit alpha 13 OEE protein 1 9 OEE protein 2 Glycolysis 14 Fructose-bisphosphate aldolase 15 Fructose-bisphosphate aldolase 18 Aldolase 21 Enolase Stress response 34 Heat shock protein STI 36 Cold shock domain protein 3 5 Pathogenesis related protein 10 Oxidation-reduction 2 Superoxide dismutase [Cu-Zn] 2 6 Glutathione transferase 7 Glutathione transferase Pyrimidine biosynthesis 28 Putative UMP/CMP kinase a
NCBI accession no.
Species
Tpi/Epi 2)
Tmw/Emw3)
Cov (%)4)
Score
Average change5)
Q9ZRA8 AAA66495 Q9ZRA8 AAB08791 ABD92936 ABD92936 ACG24494 NP_001048438 Q43470 CAA74592 CAA44259
Triticum aestivum Oryza sativa japonica Group Triticum aestivum Hordeum vulgare Triticum aestivum Triticum aestivum Zea mays Oryza sativa japonica Group Hordeum vulgare Hordeum vulgare Hordeum vulgare
4.73/5.02 4.78/5.09 4.73/5.04 4.92/5.01 4.81/5.09 4.81/5.13 5.31/5.36 4.62/4.57 4.67/4.74 4.73/4.76 4.83/4.89
50.276/49.379 48.532/50.300 50.280/52.430 49.580/52.250 49.740/50.950 49.740/50.610 41.653/55.237 29.252/31.060 29.673/34.243 29.924/33.040 29.000/28.982
70 62 70 49 48 35 25 36 56 58 41
2 194 1 768 2 194 1 068 972 786 394 487 992 818 644
-2.89 -2.18 -1.5 -3.56 -2.83 -2.31 -1.5 -1.59 -1.63 -1.61 -2.47
NP_114267 AAU11113 CAA44027 NP_114267 AAK72519 NP_114267 P08823
Triticum aestivum Psathyrostachys huashanica Triticum aestivum Triticum aestivum Lemna turionifera Triticum aestivum Triticum aestivum
6.22/5.97 6.13/4.52 6.60/5.08 6.22/6.08 6.32/5.81 6.22/5.91 4.83/4.95
52.817/11.356 52.826/14.914 46.973/21.313 52.817/29.813 49.590/26.189 52.817/27.022 57.485/61.261
23 10 17 35 17 25 42
641 442 217 525 389 502 1314
3.96 + 1.5 -4.19 1.6 + -2.32
ABQ52657 Q00434
Leymus chinensis Triticum aestivum
6.08/5.10 8.84/5.68
34.490/33.787 27.253/24.213
28 24
480 177
-5.35 -4
ACG36798 ACG36798 AAP80661 NP_001105371
Zea mays Zea mays Triticum aestivum Zea mays
7.63/5.29 7.63/5.49 8.90/5.63 5.70/5.40
41.752/39.331 41.752/38.290 23.183/28.807 48.132/54.588
16 16 22 28
324 295 315 413
-2.17 -2.66 1.82 2.37
ACG45057 BAD08701 ACG68733
Zea mays Triticum aestivum Triticum aestivum
6.26/5.88 5.73/5.80 5.19/5.09
65.506/81.849 21.530/21.611 17.053/12.520
16 60 51
190 451 465
-3.4 9.26
NP_001060564 CAC94005 CAC94005
Oryza sativa japonica Group Triticum aestivum Triticum aestivum
5.92/5.92 5.01/5.10 5.01/5.01
15.071/13.385 26.395/26.189 26.395/26.543
19 33 21
120 503 296
-4.04 3.4 1.91
NP_001048295
Oryza sativa japonica Group
5.16/5.22
23.541/22.904
42
634
-1.88
1)
Protein spot number in the gel. Tpi, pI value calculated using the amino acid sequence; Epi, pI value as estimated by the migration of the protein through 2-DE gel. 3) Tmw, Mr calculated using the amino acid sequence; Emw, Mr as estimated by the migration of the protein through 2-DE gel. 4) Percentage of predicted peptide sequences covered by the matched peptide sequences from the MS/MS peak list; protein coverage was >15%. 5) Protein abundance expressed as an average of the relative volumes of the spots from the inhibited tiller samples divided by the relative volumes of the spots from the normal tiller samples, up-regulated (+v) or down-regulated (-), absent in normal tiller samples and inhibited tiller samples. The changes in relative volume were statistically significant (P<0.05). 2)
expression of PCNA in the normal tillers is 1.6-fold higher than that in the spike-development inhibited tillers and thus exhibits the same expression pattern as mentioned for DEPs above. In the present study, three 143-3 proteins (spots 30, 31 and 32), which are involved in multiple biological processes, including hormone signal transduction, cell cycle regulation, and light signal response, were also identified and determined to be down-regulated. These protein expression changes reflected the decreased cell division and proliferation correlated with the phenotype of spike inhibition.
Therefore, cell division and proliferation may have stopped in the inhibited tillers.
RuBisCo large subunits (RLS) were degraded and located at different positions on the 2-DE gel Compared with the theoretical Mr value, the experimental values predicted by SDS-PAGE have a deviation of approximately ±10% (Wan and Liu 2008). In the category of proteins involved in photosynthesis, six spots were identified as RLS (spots 1, 11, 16, 33, 37, and
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ZHENG Yong-sheng et al.
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40). The experimental masses of these RLS spots were significantly less than the theoretical values (Table 2). Five of the six spots were significantly up-regulated, while spot 33 was down-regulated. Spot 11 was absent from gels of the normal tillers (Fig. 3-C). In addition, RLSbinding protein subunit alpha (RLS-b, spot 20), OEE protein 1 (spot 13), and OEE protein 2 (spot 9) were also identified and found to be down-regulated in inhibited tillers. Their theoretical masses were equal to the experimental values; therefore, they were intact proteins. The degradation of RLS indicates that physiological processes might have been impaired and that the photosynthetic efficiency has dropped in inhibited tillers. Four DEPs (spots 14, 15, 18, and 21) were identified as being involved in glycolysis. Two DEPs (spots 14 and 15) were isoforms of fructose-bisphosphate aldolase (CpFBA), located in the chloroplasts. These DEPs were both down-regulated and exhibited the same expression pattern as the three photosynthetic proteins described above. One enolase (spot 21), located in the cytoplasm, was significantly up-regulated. It exhibited a 2-fold change compared with enolase in normal tillers. Enolase is a rate-limiting enzyme in glycolysis and plays an important role in energy metabolism.
DEPs involved in stress response and oxidation stress changed dramatically Three DEPs, including pathogenesis-related protein 10 (PR10, spot 5), heat shock protein STI (spot 34), and cold shock domain protein 3 (spot 36), changed dramatically. The amounts of PR10 increased 9-fold in the inhibited tillers. PR10 can be induced by cold temperatures, salt, copper stress, and other related oxidative stresses (36-38). In the oxidation-reduction category, one superoxide dismutase (SOD, spot 2) and two isoforms of GSTs were identified. The SOD was down-regulated, and two GSTs were up-regulated in normal tillers. SOD is located in the cytoplasm and provides the first line of defense against oxygen toxicity by catalyzing the reduction of O 2 to yield H2O 2 (Carlioz and Touati 1986; Orr and Sohal 1994). Plant GSTs are key enzymes that defend against xenobiotic toxicity (Rakwal et al. 1999; Cui et al. 2005). The down-regulation of SOD might stifle the removal of oxygen radicals and lead to oxidative stress, which is
consistent with the downstream up-regulation of GSTs in response to oxidation stress.
DISCUSSION In the present study, one wheat mutant that exhibited normal tillering capacity but could not develop spikes was reported and used in the investigation of the relevant genetic mechanism by 2-DE and MS/MS. The tillers in 3558 line were classified into two groups: normal that can develop spikes, and inhibited that can’t develop spikes. The genetic background of the normal sample was completely consistent with that of the inhibited sample. Therefore, the DEPs identified using 2-DE and MS/MS reflected real biological differences and were closely correlated with the spike-development phenotype. These DEPs and their corresponding metabolic pathways are important to the study of spike development in wheat. Using proteomics techniques, we identified DEPs, including 14-3-3 proteins, PCNA, and tubulin. Using morphological and anatomical analyses, we found that the stems of inhibited tillers could not join and exhibited a dwarf phenotype. 14-3-3 proteins can bind REPRESSION OF SHOOT GROWTH (RSG, a transcriptional activator) and are involved in gibberellins (GAs) homeostasis (Ishida et al. 2004, 2008). 14-3-3 proteins can also interact with the BRI1 EMS SUPPRESSOR1 (BES1) and BRASSINAZOLE RESISTANT1 (BZR1) and are involved in Brassinosteroids (BRs) signal transduction to regulate plant growth and development (Ryu et al. 2010). 14-3-3 proteins can control the K+ channel activity and are involved in abscisic acid (ABA) signal transduction (Wijngaard et al. 2005). In the present study, three 14-3-3 proteins were observed to be downregulated in inhibited tillers. These 14-3-3 proteins were involved in the stem elongation and spike-development process in wheat. The expression patterns of these 14-3-3 proteins were closely related to the spike-development inhibition phenotype. The down-regulation of 14-3-3 proteins suggested that the hormone signaling pathways might be blocked and was related to the phenotype that could not joint. 14-3-3 proteins can also interact with the proteins that regulate cell cycle and thus influence cell division and proliferation (Pignocchi et al. 2009; Pignocchi and
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd.
Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558
Doonan 2011). PCNA, a cell cycle marker protein, is used to evaluate cell proliferation indicators, and changes in its expression are closely correlated with DNA synthesis (Gary et al. 1997). PCNA is essential for chromosomal DNA replication as a DNA sliding clamp for DNA polymerase delta. Six alpha and beta tubulins were also observed to be down-regulated in inhibited tillers. In addition, our unpublished data reveal some cycle-regulation genes that were also downregulated in the gene chip analysis. Therefore, the development process of the inhibited tillers is blocked, and the tillers cannot develop into spikes. The observed changes in the protein levels were consistent with the spike-development inhibition phenotype. Multiple spots were identified as RuBisCO large subunits. A comparison of the theoretical and experimental Mr values confirmed that these RuBisCO large subunits were degraded, while most of RuBisCO large subunits were up-regulated in the inhibited tillers. Degradation of RuBisCO large subunits has also been observed in chilled rice seedling leaves and cold-stressed rice seedlings (Yan et al. 2006; Wan and Liu 2008). The intact DEPs involved in photosynthesis were all observed to be down-regulated in inhibited tillers. These results demonstrate that physiological processes and photosynthetic capability decreased in inhibited tillers relative to normal tillers. To maintain normal metabolism, the inhibited tiller cells were forced to up-regulate the glycolytic metabolic pathway to obtain a large amount of energy. Thus, energy shortage might be one of the important reasons that the inhibited tillers cannot develop into normal tillers. These DEPs and related metabolic pathways may be important for understanding the mechanism of spikedevelopment inhibition. Our results provided a theoretical basis for studying spike development.
CONCLUSION To understand the molecular mechanism of spike-development inhibition, we identified proteins and metabolic pathways related to a spike-development inhibition phenotype. The spike-development inhibited tillers of wheat line 3558 stopped cell division and growth and remained at the elongation stage until the end of their life. DEPs involved in cell cycle regulation, such
403
as 14-3-3 protein, PCNA, and tubulin, were identified and found to be closely correlated with the inhibition phenotype. In addition, six degraded RuBisCO large subunits were found in different locations on the 2-DE gels, and most of these large subunits were up-regulated in inhibited tillers. These DEPs and related metabolic pathways might be important to the spike-development inhibition mechanism and provide a foundation for studying the wheat-development process.
MATERIALS AND METHODS Materials The wheat mutant line 3558 (T. aestivum L., 2n=6x=42, AABBDD) is the seventh-generation progeny of a cross between the wheat 4783 and Youla (T. aestivum L., 2n=6x=42, AABBDD), both of which are Agropyron cristatum-derived lines from the cross between Fukuhokomugi (T. aestivum L., 2n=6x=42, AABBDD) and Z559 (A. cristatum L. Gaertn. 2n=4x=28, PPPP; GenBank accession no.: Z559). This mutant line was a generous gift from the Institute of Crop Science , Chinese Aceademy of Agricultural Sciecnes, Beijing, China.
Protein extraction and quantification, twodimensional electrophoresis (2-DE) analysis, gel staining, image analysis, and in-gel digestion The normal tillers which developed into spikes and spikedevelopment inhibition tillers in the 3558 line at the elongation stage were classified into two groups and used for protein extraction (Fig. 1-B). Three replicates were used for each group. Proteins were extracted according to a previously published method (Shen et al. 2009) and quantified using 2-D Quant Kit Reagents (GE Healthcare, USA) with bovine serum albumin as the calibration standard. 2-DE analysis was conducted according to a previously published method (Wan and Liu 2008) based on isoelectric focusing (IEF) with an IPGphor IEF System and SDS PAGE in an Ettan DALT System (GE Healthcare, USA). Protein spots were detected using a modified colloidal Coomassie brilliant blue R-350 stain (GE Healthcare, USA). The 2-DE gels were fixed in 40% (v/v) ethanol/10% (v/v) acetic acid for 30 min and equilibrated in 10% (v/v) acetic acid for 20 min. Next, the gels were stained for 10 min at 90-100°C in dye liquor (one R-350 tablet dissolved in 1.6 L of 10% acetic acid) and then rinsed with 10% (v/v) acetic acid until the gel spots were distinct from the background. The gels were stored in distilled water. Gel images were acquired using a Labscan 3.0 densitometer (GE Healthcare, USA).
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ImageMaster 2D platinum ver. 6.0 software (GE Healthcare, USA) was used to analyze the 2-DE gel. The spots were detected automatically with the following parameter settings: smooth, 6; minimum area, 65; and saliency, 30. Proteins were considered significantly differentially expressed when their relative spot volumes from the normal and inhibited tillers differed by more than ±1.5-fold (Student’s t-test, P<0.05) (Wan and Liu 2008). Differentially expressed protein spots were subjected to tryptic digestion according to a previous method (Yao et al. 2006).
Protein identification by Nano LC-HDMS MS/MS and database searching The protein spots were identified by Nano LC-high-definition MS (HDMS)/MS/MS performed using a nanoAcquity UPLC System (Waters Corp., Milford, USA) and a Synapt HDMS with a nanospray ion source (Waters Corp., Milford, USA) which was operated in the data-dependent mode for the MS/MS scans (2 s per scan). Tandem mass spectra were acquired for the two most intense peaks from each MS scan (1 s per scan) in the positive ion mode. The voltage of the non-coated capillary was 2 300 V. Glu-fibrinopeptide served as the calibration standard for the MS/ MS mode. Peak lists for each LC-MS/MS run were generated using PLGS 2.2 software (Waters) and automatically combined into a single PKL file. MS/MS ion database searches were performed using the Mascot web-based service (http://www.matrixscience.com). The Viridiplantae database from NCBInr was specified as the search database, and identification occurred when extensive similarity between a sample sequence and one in the database was observed (P<0.05). When protein isoforms were observed, the presence of each protein isoform was confirmed by the identification of at least two unique peptides (Wan and Liu 2008).
Acknowledgements We thank Prof. Wang Baichen from the Institute of Botany, Chinese Academy of Sciences, for help with the analysis of proteomic data and the revision of this article, Prof. Liu Jinyuan from Tsinghua University, China, for assisting with the design and analysis of the 2-D PAGE experiment, and Wang Hongxia and Li Weihua from the Military Medical Science Academy of the People’s Liberation Army for assisting with the nano LC-HDMS MS MS/MS study. This work was supported by the National High Technology Research and Development Program of China (2011AA100102 and 2006AA10Z174).
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(Managing editor SUN Lu-juan)
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March 2013
2013, 12(3): 398-405
RESEARCH ARTICLE
Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558 ZHENG Yong-sheng1, MA Xiao-gang2, CHI De-zhao2, GAO Ai-nong1, LI Li-hui1 and LIU Wei-hua1 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China 2 Qinghai Academy of Agriculture and Forestry, Xining 810000, P.R.China 1
Abstract Spike number is one of three yield-related factors and is closely related to wheat yield. In the present study, we found that the inhibited and normal tillers of the 3558 line presented phenotypic differences at the elongation stage by morphological and anatomical analysis. We then initiated a proteomic study using two-dimensional electrophoresis (2-DE) and nanoscale liquid chromatography-high-definition tandem mass spectroscopy, to isolate and identify the key proteins and metabolic pathways related to spike-development inhibition. A total of 31 differentially expressed proteins (DEPs), which were mainly involved in cell cycle regulation, photosynthesis, glycolysis, stress response, and oxidation-reduction reactions, were isolated and identified. 14-3-3-like proteins and proliferating cell nuclear antigen (PCNA), involved in cell-cycle regulation, were dramatically down-regulated in inhibited tillers compared to normal tillers. Six spots corresponding to degraded Rubisco large subunits, involved in photosynthesis, were detected in different locations of the 2-DE gels and were up-regulated in inhibited tillers. In addition, the relative levels of DEPs involved in glycolysis and oxidationreduction reactions changed dramatically. Development was blocked or delayed at the elongation stage in the inhibited tillers of 3558. Weakened energy metabolism might be one reason that the inhibited tillers could not joint and develop into spikes. These DEPs and related metabolic pathways are significant for understanding the mechanism of spike-development inhibition and studying the spike-development process in wheat. Key words: wheat, spike-development inhibition, 2-DE, differentially expressed proteins
INTRODUCTION The problem of food security has gained attention because of the continued growth of the population, substantial reduction of agricultural land, climate change, and frequent pest and disease occurrences. Wheat is one of the most important food crops and plays an irreplaceable role in improving and stabilizing food production to address food security issues. In recent years, because wheat acreage has declined and production is
at standstill, the wheat supply and demand relationship had become increasingly acute. Improving wheat yield through genetic alteration has become one of the most prominent areas of study for wheat breeders and scientists. Panicles (spikes) per unit area, number of spikelets per panicle and grain weight are three important yield components for crop plants. The number of spikes per unit area has been found to be the most important component of yield, accounting for 87% of the variation in yield (Gravois and McNew 1993). Highyield breeding should therefore focus on increasing the
Received 2 May, 2012 Accepted 24 September, 2012 ZHENG Yong-sheng, Tel: +86-531-83178713, E-mail: [email protected]; Correspondance LIU Wei-hua, Tel: +86-10-62176077, Fax: +86-10-62189650, E-mail: [email protected] © 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60239-7
Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558
number of spikes per unit area. Tillering in cereal crops such as rice and wheat involves two stages: the formation of axillary buds and the subsequent outgrowth of the buds (Hanada 1993). The formation of axillary buds determines the tillering capacity and is potentially controlled by a single gene, while the development of the buds into spikes is a complex developmental process (Li et al. 2003). In bread wheat, the genetic mechanisms of tillering capacity have been explored. Richards (1988) reported a low-tillering wheat mutation called Israel uniculm-492, which was controlled by one recessive gene, tin; tiller formation was also blocked or affected by high temperature, day length, and extreme cultivation conditions (Richards 1988). The recessive gene has been mapped to the short arm of chromosome 1A (Spielmeyer and Richards 2004). A dominant gene, tin2, which controls the oligoculm phenotype, was located on chromosome 2A as determined by the genetic analysis of the Chinese Spring (CS) monosomic series (Peng et al. 2007). Kuraparthy et al. (2007) isolated a wheat mutant that produced one main culm and lost tillering capability using ethyl methanesulphonate (EMS)-based mutagenesis in diploid wheat. The mutant single recessive gene tin3 was located on the long arm of chromosome 3A and was found to be closely linked with Xpsr1205 (Kuraparthy et al. 2007). Although the wheat genes influencing the formation of axillary buds have been described, the mechanism of tiller development into spikes in wheat is largely uncharacterized. The wheat spike-inhibition phenotype of 3558 is controlled by a single recessive gene which was termed the fertile tiller inhibition gene and was mapped to chromo-
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some 1AS (Zhang et al. 2012). In the present study, the wheat mutant line 3558, which has normal tillering capacity (Fig. 1-A) but a spike-development inhibition phenotype (Fig. 1-C), was used to analyze the key proteins and metabolic pathways correlated with spike development using two-dimensional electrophoresis (2-DE) and nano-scale liquid chromatography-high-definition tandem mass spectroscopy. Our study will increase our understanding of spike development in wheat and provide a genetic basis to improve wheat yield.
RESULTS 2-DE mapping, protein spot identification and functional classification To delineate the key DEPs and metabolic pathways underlying the phenotypic differences between normal and inhibited tillers of 3558 line, we analyzed their proteomes at the elongation stage (Fig. 1-B). A total of (1 500±50) spots were reproducibly detected in the three gel replicates (Fig. 2). For the inhibited tillers, 35 spots had significantly altered intensities compared with the corresponding normal tillers (P<0.05), and the intensities for these spots increased by more than 1.5 fold. For the inhibited tillers, 13 spots were significantly upregulated and 22 were down-regulated. A total of 35 differentially expressed protein (DEPs) spots were analyzed by NanoLC-HDMS MS/MS, and 31 spots were successfully identified (Table). The 31 DEPs were grouped into 6 categories according to their functional annotations in the UniProtKB
Fig. 1 Morphology and anatomy of line 3558. A, seedling morphology of line 3558 at the elongation stage. Arrows indicate an inhibited tiller (1) and a normal tiller (2). B, the anatomies of inhibited (left) and normal (right) tillers. C, the morphologies of line 3558.
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Fig. 2 2-DE reference maps of normal and inhibited tillers at the elongation stage. A, 2-DE map of the normal tiller proteome. B, 2-DE map of the inhibited tiller proteome. C, key DEP spots.
database. They were mainly involved in cell cycle regulation (11 spots), photosynthesis (9 spots), glycolysis (4 spots), stress response (3 spots), and oxidation-reduction reactions (3 spots, Table 1).
DEPs involved in cell cycle regulation were all down-regulated Eleven of the identified DEPs were involved in cell cycle regulation. They were all down-regulated in the spikedevelopment inhibition tillers. Among these DEPs, six were alpha and beta subunits of tubulin: two isoforms of the tubulin beta-5 subunit (spots 50 and 56), two
isoforms of the alpha tubulin-4D subunit (spots 58 and 59), one tubulin beta subunit (spot 52), and one tubulin alpha subunit (spot 57). One additional spot, identified as actin-2, was also down-regulated. Tubulin and actin form the microtubules and microfilament, respectively, and constitute the cell cytoskeleton. They have important roles in maintaining the cell’s form and the process of cell division. Proliferating cell nuclear antigen (PCNA, spot 92) was drastically down-regulated in the inhibited tillers. This protein is a DNA polymerase accessory factor required for DNA replication, and its expression is an indicator of cell proliferation (Gary et al. 1997). The
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Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558
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Table Differentially expressed proteins in the normal and inhibited tillers of the 3558 line at the elongation stage Spot no. 1)
Protein name
Cell structure and cell cycle regulation 50 Tubulin beta-5 chain 52 Beta-tubulin 56 Tubulin beta-5 chain 57 Alpha tubulin 58 Alpha tubulin-4D 59 Alpha tubulin-4D 23 Actin-2 53 Proliferating cell nuclear antigen 30 14-3-3-like protein B 31 14-3-3 protein 32 14-3-3 protein homologue Photosynthesis 1 RuBisCO large subunit 11 RuBisCO large subunit 16 RuBisCO large subunit 33 RuBisCO large subunit 37 RuBisCO large subunit 40 RuBisCO large subunit 20 RuBisCO large subunit-binding protein subunit alpha 13 OEE protein 1 9 OEE protein 2 Glycolysis 14 Fructose-bisphosphate aldolase 15 Fructose-bisphosphate aldolase 18 Aldolase 21 Enolase Stress response 34 Heat shock protein STI 36 Cold shock domain protein 3 5 Pathogenesis related protein 10 Oxidation-reduction 2 Superoxide dismutase [Cu-Zn] 2 6 Glutathione transferase 7 Glutathione transferase Pyrimidine biosynthesis 28 Putative UMP/CMP kinase a
NCBI accession no.
Species
Tpi/Epi 2)
Tmw/Emw3)
Cov (%)4)
Score
Average change5)
Q9ZRA8 AAA66495 Q9ZRA8 AAB08791 ABD92936 ABD92936 ACG24494 NP_001048438 Q43470 CAA74592 CAA44259
Triticum aestivum Oryza sativa japonica Group Triticum aestivum Hordeum vulgare Triticum aestivum Triticum aestivum Zea mays Oryza sativa japonica Group Hordeum vulgare Hordeum vulgare Hordeum vulgare
4.73/5.02 4.78/5.09 4.73/5.04 4.92/5.01 4.81/5.09 4.81/5.13 5.31/5.36 4.62/4.57 4.67/4.74 4.73/4.76 4.83/4.89
50.276/49.379 48.532/50.300 50.280/52.430 49.580/52.250 49.740/50.950 49.740/50.610 41.653/55.237 29.252/31.060 29.673/34.243 29.924/33.040 29.000/28.982
70 62 70 49 48 35 25 36 56 58 41
2 194 1 768 2 194 1 068 972 786 394 487 992 818 644
-2.89 -2.18 -1.5 -3.56 -2.83 -2.31 -1.5 -1.59 -1.63 -1.61 -2.47
NP_114267 AAU11113 CAA44027 NP_114267 AAK72519 NP_114267 P08823
Triticum aestivum Psathyrostachys huashanica Triticum aestivum Triticum aestivum Lemna turionifera Triticum aestivum Triticum aestivum
6.22/5.97 6.13/4.52 6.60/5.08 6.22/6.08 6.32/5.81 6.22/5.91 4.83/4.95
52.817/11.356 52.826/14.914 46.973/21.313 52.817/29.813 49.590/26.189 52.817/27.022 57.485/61.261
23 10 17 35 17 25 42
641 442 217 525 389 502 1314
3.96 + 1.5 -4.19 1.6 + -2.32
ABQ52657 Q00434
Leymus chinensis Triticum aestivum
6.08/5.10 8.84/5.68
34.490/33.787 27.253/24.213
28 24
480 177
-5.35 -4
ACG36798 ACG36798 AAP80661 NP_001105371
Zea mays Zea mays Triticum aestivum Zea mays
7.63/5.29 7.63/5.49 8.90/5.63 5.70/5.40
41.752/39.331 41.752/38.290 23.183/28.807 48.132/54.588
16 16 22 28
324 295 315 413
-2.17 -2.66 1.82 2.37
ACG45057 BAD08701 ACG68733
Zea mays Triticum aestivum Triticum aestivum
6.26/5.88 5.73/5.80 5.19/5.09
65.506/81.849 21.530/21.611 17.053/12.520
16 60 51
190 451 465
-3.4 9.26
NP_001060564 CAC94005 CAC94005
Oryza sativa japonica Group Triticum aestivum Triticum aestivum
5.92/5.92 5.01/5.10 5.01/5.01
15.071/13.385 26.395/26.189 26.395/26.543
19 33 21
120 503 296
-4.04 3.4 1.91
NP_001048295
Oryza sativa japonica Group
5.16/5.22
23.541/22.904
42
634
-1.88
1)
Protein spot number in the gel. Tpi, pI value calculated using the amino acid sequence; Epi, pI value as estimated by the migration of the protein through 2-DE gel. 3) Tmw, Mr calculated using the amino acid sequence; Emw, Mr as estimated by the migration of the protein through 2-DE gel. 4) Percentage of predicted peptide sequences covered by the matched peptide sequences from the MS/MS peak list; protein coverage was >15%. 5) Protein abundance expressed as an average of the relative volumes of the spots from the inhibited tiller samples divided by the relative volumes of the spots from the normal tiller samples, up-regulated (+v) or down-regulated (-), absent in normal tiller samples and inhibited tiller samples. The changes in relative volume were statistically significant (P<0.05). 2)
expression of PCNA in the normal tillers is 1.6-fold higher than that in the spike-development inhibited tillers and thus exhibits the same expression pattern as mentioned for DEPs above. In the present study, three 143-3 proteins (spots 30, 31 and 32), which are involved in multiple biological processes, including hormone signal transduction, cell cycle regulation, and light signal response, were also identified and determined to be down-regulated. These protein expression changes reflected the decreased cell division and proliferation correlated with the phenotype of spike inhibition.
Therefore, cell division and proliferation may have stopped in the inhibited tillers.
RuBisCo large subunits (RLS) were degraded and located at different positions on the 2-DE gel Compared with the theoretical Mr value, the experimental values predicted by SDS-PAGE have a deviation of approximately ±10% (Wan and Liu 2008). In the category of proteins involved in photosynthesis, six spots were identified as RLS (spots 1, 11, 16, 33, 37, and
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40). The experimental masses of these RLS spots were significantly less than the theoretical values (Table 2). Five of the six spots were significantly up-regulated, while spot 33 was down-regulated. Spot 11 was absent from gels of the normal tillers (Fig. 3-C). In addition, RLSbinding protein subunit alpha (RLS-b, spot 20), OEE protein 1 (spot 13), and OEE protein 2 (spot 9) were also identified and found to be down-regulated in inhibited tillers. Their theoretical masses were equal to the experimental values; therefore, they were intact proteins. The degradation of RLS indicates that physiological processes might have been impaired and that the photosynthetic efficiency has dropped in inhibited tillers. Four DEPs (spots 14, 15, 18, and 21) were identified as being involved in glycolysis. Two DEPs (spots 14 and 15) were isoforms of fructose-bisphosphate aldolase (CpFBA), located in the chloroplasts. These DEPs were both down-regulated and exhibited the same expression pattern as the three photosynthetic proteins described above. One enolase (spot 21), located in the cytoplasm, was significantly up-regulated. It exhibited a 2-fold change compared with enolase in normal tillers. Enolase is a rate-limiting enzyme in glycolysis and plays an important role in energy metabolism.
DEPs involved in stress response and oxidation stress changed dramatically Three DEPs, including pathogenesis-related protein 10 (PR10, spot 5), heat shock protein STI (spot 34), and cold shock domain protein 3 (spot 36), changed dramatically. The amounts of PR10 increased 9-fold in the inhibited tillers. PR10 can be induced by cold temperatures, salt, copper stress, and other related oxidative stresses (36-38). In the oxidation-reduction category, one superoxide dismutase (SOD, spot 2) and two isoforms of GSTs were identified. The SOD was down-regulated, and two GSTs were up-regulated in normal tillers. SOD is located in the cytoplasm and provides the first line of defense against oxygen toxicity by catalyzing the reduction of O 2 to yield H2O 2 (Carlioz and Touati 1986; Orr and Sohal 1994). Plant GSTs are key enzymes that defend against xenobiotic toxicity (Rakwal et al. 1999; Cui et al. 2005). The down-regulation of SOD might stifle the removal of oxygen radicals and lead to oxidative stress, which is
consistent with the downstream up-regulation of GSTs in response to oxidation stress.
DISCUSSION In the present study, one wheat mutant that exhibited normal tillering capacity but could not develop spikes was reported and used in the investigation of the relevant genetic mechanism by 2-DE and MS/MS. The tillers in 3558 line were classified into two groups: normal that can develop spikes, and inhibited that can’t develop spikes. The genetic background of the normal sample was completely consistent with that of the inhibited sample. Therefore, the DEPs identified using 2-DE and MS/MS reflected real biological differences and were closely correlated with the spike-development phenotype. These DEPs and their corresponding metabolic pathways are important to the study of spike development in wheat. Using proteomics techniques, we identified DEPs, including 14-3-3 proteins, PCNA, and tubulin. Using morphological and anatomical analyses, we found that the stems of inhibited tillers could not join and exhibited a dwarf phenotype. 14-3-3 proteins can bind REPRESSION OF SHOOT GROWTH (RSG, a transcriptional activator) and are involved in gibberellins (GAs) homeostasis (Ishida et al. 2004, 2008). 14-3-3 proteins can also interact with the BRI1 EMS SUPPRESSOR1 (BES1) and BRASSINAZOLE RESISTANT1 (BZR1) and are involved in Brassinosteroids (BRs) signal transduction to regulate plant growth and development (Ryu et al. 2010). 14-3-3 proteins can control the K+ channel activity and are involved in abscisic acid (ABA) signal transduction (Wijngaard et al. 2005). In the present study, three 14-3-3 proteins were observed to be downregulated in inhibited tillers. These 14-3-3 proteins were involved in the stem elongation and spike-development process in wheat. The expression patterns of these 14-3-3 proteins were closely related to the spike-development inhibition phenotype. The down-regulation of 14-3-3 proteins suggested that the hormone signaling pathways might be blocked and was related to the phenotype that could not joint. 14-3-3 proteins can also interact with the proteins that regulate cell cycle and thus influence cell division and proliferation (Pignocchi et al. 2009; Pignocchi and
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Comparative Proteomic Analysis of Spike-Development Inhibited and Normal Tillers of Wheat 3558
Doonan 2011). PCNA, a cell cycle marker protein, is used to evaluate cell proliferation indicators, and changes in its expression are closely correlated with DNA synthesis (Gary et al. 1997). PCNA is essential for chromosomal DNA replication as a DNA sliding clamp for DNA polymerase delta. Six alpha and beta tubulins were also observed to be down-regulated in inhibited tillers. In addition, our unpublished data reveal some cycle-regulation genes that were also downregulated in the gene chip analysis. Therefore, the development process of the inhibited tillers is blocked, and the tillers cannot develop into spikes. The observed changes in the protein levels were consistent with the spike-development inhibition phenotype. Multiple spots were identified as RuBisCO large subunits. A comparison of the theoretical and experimental Mr values confirmed that these RuBisCO large subunits were degraded, while most of RuBisCO large subunits were up-regulated in the inhibited tillers. Degradation of RuBisCO large subunits has also been observed in chilled rice seedling leaves and cold-stressed rice seedlings (Yan et al. 2006; Wan and Liu 2008). The intact DEPs involved in photosynthesis were all observed to be down-regulated in inhibited tillers. These results demonstrate that physiological processes and photosynthetic capability decreased in inhibited tillers relative to normal tillers. To maintain normal metabolism, the inhibited tiller cells were forced to up-regulate the glycolytic metabolic pathway to obtain a large amount of energy. Thus, energy shortage might be one of the important reasons that the inhibited tillers cannot develop into normal tillers. These DEPs and related metabolic pathways may be important for understanding the mechanism of spikedevelopment inhibition. Our results provided a theoretical basis for studying spike development.
CONCLUSION To understand the molecular mechanism of spike-development inhibition, we identified proteins and metabolic pathways related to a spike-development inhibition phenotype. The spike-development inhibited tillers of wheat line 3558 stopped cell division and growth and remained at the elongation stage until the end of their life. DEPs involved in cell cycle regulation, such
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as 14-3-3 protein, PCNA, and tubulin, were identified and found to be closely correlated with the inhibition phenotype. In addition, six degraded RuBisCO large subunits were found in different locations on the 2-DE gels, and most of these large subunits were up-regulated in inhibited tillers. These DEPs and related metabolic pathways might be important to the spike-development inhibition mechanism and provide a foundation for studying the wheat-development process.
MATERIALS AND METHODS Materials The wheat mutant line 3558 (T. aestivum L., 2n=6x=42, AABBDD) is the seventh-generation progeny of a cross between the wheat 4783 and Youla (T. aestivum L., 2n=6x=42, AABBDD), both of which are Agropyron cristatum-derived lines from the cross between Fukuhokomugi (T. aestivum L., 2n=6x=42, AABBDD) and Z559 (A. cristatum L. Gaertn. 2n=4x=28, PPPP; GenBank accession no.: Z559). This mutant line was a generous gift from the Institute of Crop Science , Chinese Aceademy of Agricultural Sciecnes, Beijing, China.
Protein extraction and quantification, twodimensional electrophoresis (2-DE) analysis, gel staining, image analysis, and in-gel digestion The normal tillers which developed into spikes and spikedevelopment inhibition tillers in the 3558 line at the elongation stage were classified into two groups and used for protein extraction (Fig. 1-B). Three replicates were used for each group. Proteins were extracted according to a previously published method (Shen et al. 2009) and quantified using 2-D Quant Kit Reagents (GE Healthcare, USA) with bovine serum albumin as the calibration standard. 2-DE analysis was conducted according to a previously published method (Wan and Liu 2008) based on isoelectric focusing (IEF) with an IPGphor IEF System and SDS PAGE in an Ettan DALT System (GE Healthcare, USA). Protein spots were detected using a modified colloidal Coomassie brilliant blue R-350 stain (GE Healthcare, USA). The 2-DE gels were fixed in 40% (v/v) ethanol/10% (v/v) acetic acid for 30 min and equilibrated in 10% (v/v) acetic acid for 20 min. Next, the gels were stained for 10 min at 90-100°C in dye liquor (one R-350 tablet dissolved in 1.6 L of 10% acetic acid) and then rinsed with 10% (v/v) acetic acid until the gel spots were distinct from the background. The gels were stored in distilled water. Gel images were acquired using a Labscan 3.0 densitometer (GE Healthcare, USA).
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ImageMaster 2D platinum ver. 6.0 software (GE Healthcare, USA) was used to analyze the 2-DE gel. The spots were detected automatically with the following parameter settings: smooth, 6; minimum area, 65; and saliency, 30. Proteins were considered significantly differentially expressed when their relative spot volumes from the normal and inhibited tillers differed by more than ±1.5-fold (Student’s t-test, P<0.05) (Wan and Liu 2008). Differentially expressed protein spots were subjected to tryptic digestion according to a previous method (Yao et al. 2006).
Protein identification by Nano LC-HDMS MS/MS and database searching The protein spots were identified by Nano LC-high-definition MS (HDMS)/MS/MS performed using a nanoAcquity UPLC System (Waters Corp., Milford, USA) and a Synapt HDMS with a nanospray ion source (Waters Corp., Milford, USA) which was operated in the data-dependent mode for the MS/MS scans (2 s per scan). Tandem mass spectra were acquired for the two most intense peaks from each MS scan (1 s per scan) in the positive ion mode. The voltage of the non-coated capillary was 2 300 V. Glu-fibrinopeptide served as the calibration standard for the MS/ MS mode. Peak lists for each LC-MS/MS run were generated using PLGS 2.2 software (Waters) and automatically combined into a single PKL file. MS/MS ion database searches were performed using the Mascot web-based service (http://www.matrixscience.com). The Viridiplantae database from NCBInr was specified as the search database, and identification occurred when extensive similarity between a sample sequence and one in the database was observed (P<0.05). When protein isoforms were observed, the presence of each protein isoform was confirmed by the identification of at least two unique peptides (Wan and Liu 2008).
Acknowledgements We thank Prof. Wang Baichen from the Institute of Botany, Chinese Academy of Sciences, for help with the analysis of proteomic data and the revision of this article, Prof. Liu Jinyuan from Tsinghua University, China, for assisting with the design and analysis of the 2-D PAGE experiment, and Wang Hongxia and Li Weihua from the Military Medical Science Academy of the People’s Liberation Army for assisting with the nano LC-HDMS MS MS/MS study. This work was supported by the National High Technology Research and Development Program of China (2011AA100102 and 2006AA10Z174).
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(Managing editor SUN Lu-juan)
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