Article pubs.acs.org/jpr
Proteomic Analysis of Salt Tolerance in Sugar Beet Monosomic Addition Line M14 Le Yang,†,‡ Yanjun Zhang,§,∥ Ning Zhu,∥ Jin Koh,∥ Chunquan Ma,†,‡ Yu Pan,†,‡ Bing Yu,†,‡ Sixue Chen,*,∥,†,‡ and Haiying Li*,†,‡ †
Key Laboratory of Molecular Biology of Heilongjiang Province, College of Life Sciences, Heilongjiang University, Harbin 150080, China ‡ Engineering Research Center of Agricultural Microbiology Technology, Ministry of Education, Heilongjiang University, Harbin 150500, China § Information Science and Technology School, Heilongjiang University, Harbin 150080, China ∥ Department of Biology, Genetics Institute, Plant Molecular and Cellular Biology Program, Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida 32610, United States S Supporting Information *
ABSTRACT: Understanding the mechanisms of plant salinity tolerance can facilitate plant engineering for enhanced salt stress tolerance. Sugar beet monosomic addition line M14 obtained from the intercross between Beta vulgaris L. and Beta corollif lora Zoss exhibits tolerance to salt stress. Here we report the salt-responsive characteristics of the M14 plants under 0, 200, and 400 mM NaCl conditions using quantitative proteomics approaches. Proteins from control and the salt treated M14 plants were separated using 2D-DIGE. Eighty-six protein spots representing 67 unique proteins in leaves and 22 protein spots representing 22 unique proteins in roots were identified. In addition, iTRAQ LC−MS/MS was employed to identify and quantify differentially expressed proteins under salt stress. Seventy-five differentially expressed proteins in leaves and 43 differentially expressed proteins in roots were identified. The proteins were mainly involved in photosynthesis, energy, metabolism, protein folding and degradation, and stress and defense. Moreover, gene transcription data obtained from the same samples were compared to the corresponding protein data. Thirteen proteins in leaves and 12 in roots showed significant correlation in gene expression and protein levels. These results suggest the important processes for the M14 tolerance to salt stress include enhancement of photosynthesis and energy metabolism, accumulation of osmolyte and antioxidant enzymes, and regulation of methionine metabolism and ion uptake/exclusion. KEYWORDS: sugar beet M14, 2D-DIGE, iTRAQ, RNA sequencing, salt tolerance
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INTRODUCTION
Although individual salt-responsive genes are important, plant salt tolerance is controlled by sophisticated signaling and metabolic networks. Compared with glycophytes, halophytes have developed unique structures that allow them to grow under high salt stress conditions.7 To understand salt tolerance networks, high-throughput transcriptomic and proteomic studies have been carried out. At least 2300 ESTs/cDNAs in several halophytes (e.g., Thellungiella halophila,6 Suaeda salsa,8 Aeluropus littoralis,9 Salicornia brachiata,10 and Festuca rubra ssp. Litoralis.11) exhibited differential expression under salinity conditions. Proteomic studies have revealed more than 218 salt responsive proteins in some halophytes (e.g., Salicornia europaea,12 Suaeda aegyptiaca,13 Bruguiera gymnorhiza,14 T.
Salt stress is one of the most serious threats to crop production.1,2 Soil salinization may lead to the loss of nearly half of the irrigated land by 2050.3 Studying salt tolerance mechanisms has been a focus in plant biology research.2,4 In general, high salt concentration can lead to ion imbalance, hyperosmotic stress and oxidative damage that will result in plant growth retardation, wilting or death.5 To survive salt stress, plants have evolved complex mechanisms allowing for adaptation, including selective ion uptake and exclusion, compartmentalization of Na+ in vacuoles, synthesis of compatible solutes, e.g., glycine betaine (GB) and proline, adjustment of photosynthesis, and detoxification of reactive oxygen species (ROS). Some of the salt-responsive genes involved in membrane transport, signal transduction, redox reaction and other processes have been cloned and characterized.6 © XXXX American Chemical Society
Special Issue: Agricultural and Environmental Proteomics Received: February 26, 2013
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halophila15 and Aster tripolium.16). These studies have provided important insights into the mechanisms underlying plant salt response and tolerance. To date, scarce proteomic studies in sugar beet under salt stress have been reported. Wakeel A et al.17 identified six proteins from sugar beet shoots and three proteins from roots that significantly changed under 125 mM salt, but these proteins could not be attributed to the adaptation under salt stress. Sugar beet monosomic addition line M14 was obtained from the intercross between Beta vulgaris L. and Beta corollif lora Zoss. It contains the Beta vulgaris L. genome with the addition of chromosome 9 of Beta corollif lora Zoss.18 The M14 line has exhibited interesting phenotypes, such as apomixis and tolerance to drought, cold and salt.19,20 Our previous studies have shown that M14 can tolerate 500 mM NaCl treatment, under which 38 unique proteins from leaves and 29 unique proteins from roots exhibited significant changes.21 Proteins involved in metabolism, protein folding and degradation, and photosynthesis play important roles in surviving the high salt treatment. In addition, S-adenosyl-L-methionine synthase (SAMS) was increased at both transcriptional and translational levels under high salt stress.21 In spite of the progress, a comprehensive description of the proteome changes and gene transcription in the M14 under moderate salt stress is lacking. Quantitative proteomics approaches including two-dimensional difference gel electrophoresis (2D-DIGE) and gel-free isobaric tag for relative and absolute quantification (iTRAQ) have shown complementary utility in quantifying protein changes in different biological samples.15,21 In addition to proteome level technologies, next generation sequencing has enabled high throughput genome-wide studies and has been used to explore gene expression profiles.22,23 Here we report the analyses of salt-responsive proteins in the M14 plants under 0, 200, and 400 mM NaCl conditions. Proteins from control and salt-treated M14 leaves and roots were separated using 2DDIGE. Differentially expressed proteins were identified using nanoflow liquid chromatography (LC)−MS/MS and Mascot database searching. In addition, iTRAQ LC−MS/MS was employed to identify and quantify differentially expressed proteins in the M14 salinity responses. Furthermore, we compared proteomic and transcriptional level changes under different salt stress conditions. In leaves under salt stress, proteins involved in photosynthesis, respiration, antioxidant system, methionine metabolism, and GB synthesis all increased in levels. In roots, similar changes were observed. Some of the changes are very different from the results obtained from the 500 mM NaCl stress treatment,21 indicating different mechanisms underlying M14 tolerance to different levels of salt stress.
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inhibited when the NaCl concentration increased to over 200 mM, and some M14 seedlings started to die at over 500 mM NaCl, indicating 500 mM NaCl is a high concentration that M14 can survive.21 Therefore, three NaCl concentrations were applied: 0 (control), 200 mM and 400 mM. To avoid osmotic shock, salt was gradually increased by 50 mM each day until the desired concentration was reached.24 Culturing solutions were changed daily for maintaining a stable NaCl concentration in the Hoagland solution. Leaves and roots of control and treated M14 seedlings were harvested directly into liquid nitrogen after seven days of treatment and stored in −80 °C. At least three independent biological replicates of control samples and three of treated samples were conducted for all the experiments. Protein Extraction, Quantification, and 2D-DIGE
Total leaf and root proteins were precipitated with 10% (w/v) trichloroacetic acid (TCA) in acetone at −20 °C overnight. After centrifugation at 40000g at 4 °C for 1 h, the pellets were washed three times with 80% cold acetone. The pellets were solubilized in a DIGE buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS and 30 mM Tris-Cl at room temperature for 1 h before centrifugation at 100000g at 4 °C for 1 h. The supernatants were collected and cleaned up using a 2D Cleanup kit according to the manufacturer’s instructions (GE Healthcare, USA). Total protein was quantified using a 2D Quant kit (GE Healthcare, USA). To facilitate statistical analysis, the experimental design in Table S1 (Supporting Information) was followed. Proteins were labeled using a DIGE fluor minimal labeling kit according to the manufacturer’s instructions (GE Healthcare, USA). Each dye was reconstituted in fresh N,N-dimethylformamide. Protein samples, each of 50 μg, were labeled with 400 pmol of dye at pH 8.5. Control and salt-stressed samples were labeled with either Cy3 or Cy5 dye. Equal aliquots of proteins extracted from 10 leaf or 10 root samples were mixed as the internal standard and then labeled with Cy2. The labeling reaction took place on ice in the dark for 40 min and was terminated by addition of 1 μL of 10 mM lysine. Gel electrophoresis and image analysis were conducted as previously described.21 Briefly, in the first analysis step, gel spots were detected with background subtraction, then a differential in-gel analysis (DIA) module was used to utilize the internal standard to aid spot matching between samples, and to generate a ratio of protein abundance between the proteins of the internal standard and each sample. Since the internal standard was included in each gel, this allowed normalization of all the data. In the second analysis step, biological variation (BVA) module was used to match multiples images from different gels and to provide statistical analysis of quantitative comparison of spot volumes between all the samples. Spots exhibiting more than 1.5-fold changes in salt-treated samples versus control samples and pvalues smaller than 0.05 were considered to be differentially expressed.
MATERIALS AND METHODS
Plant Materials and NaCl Treatment
In-Gel Digestion and Protein Identification
The M14 seeds were sterilized with 70% (v/v) ethanol, 0.1% (w/w) mercury chloride and 0.2% (w/w) thiram, and then sown in vermiculite for germination and watered daily. After one week, seedlings were transferred to hydroponic containers containing Hoagland solution.24 Seedlings were grown in a growth chamber with a 13 h light/11 h dark cycle, 25/20 °C day/night temperature, 450 μmol m−2 s−1 light intensity and a relative humidity of 70%. Salt treatment was initiated three weeks after sowing. On the basis of our previous studies, the M14 growth was not affected under 100 mM NaCl but
Protein in-gel tryptic digestion and nanoESI MS/MS analysis were carried out on a QSTAR XL MS/MS system (AB Sciex Inc., USA) as previously described.25 The peptide MS/MS spectra were searched against a NCBI green plant database (downloaded on October 3, 2012 with 15427932 sequences) using a Mascot search engine (Matrix Sciences Inc., U.K.). Mascot was set up to consider trypsin digestion, one miscleavage, iodoacetamide derivatization of Cys as fixed modification, and deamidation of Asn/Gln and oxidation of B
dx.doi.org/10.1021/pr400177m | J. Proteome Res. XXXX, XXX, XXX−XXX
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Figure 1. 2D-DIGE gels of leaf protein samples from plants grown in different salt stress conditions. (A) Control; (B) 200 mM NaCl; and (C) 400 mM NaCl. A total of 127 spots with significant volume changes under salt stress were labeled in (A) using the DeCyder software.
Western Blot Analysis
Met as variable modifications. The mass tolerance for parent ion was set at 0.5 Da, and that for fragment ion was set at 0.2 Da. Unambiguous identification was judged by the number of peptides (at least one peptide with more than 95% confidence), sequence coverage, the overall Mascot score and peptide ion score (p < 0.05).
Primary antibodies raised in rabbits against maize oxygen evolving enhancer protein (OEP), phosphoribulokinase (PRK), enolase, SAMS and Arabidopsis glutathione S-transferase (GST) were purchased from Huada Biotechnology Co., Ltd. (Guangzhou, China). Eighty micrograms of leaf and root proteins were subjected to 10% SDS−PAGE and electrotransferred to a polyvinylidene difluoride membrane at 100 V for 60 min. The membrane was blocked with 5% nonfat milk in a solution containing 0.2 M TRIS-HCl pH 7.6, 1.37 M NaCl, and 0.1% Tween-20 (TTBS) for 1 h at room temperature. The proteins were probed with different polyclonal antibodies (OEP, PRK, enolase, SAMS and GST at 1:1000) 3 h at room temperature, followed by three washes (each 5 min) in the TTBS solution. The membrane was then incubated with a horseradish peroxidase-conjugated goat antirabbit antibody for 1 h at room temperature, followed by three washes (each 5 min) in the TTBS solution. The blot was developed with a SuperECL Plus kit (Applygen, Beijing, China).
iTRAQ Labeling, 2D LC−MS/MS and Data Analysis
The acetone pellets from three control samples, three 200 mM NaCl treated and three 400 mM NaCl treated samples (each of 100 μg of protein) from leaves and roots were dissolved in 1% SDS, 100 mM triethylammonium bicarbonate, pH 8.5. The samples were reduced, alkylated with methylmethanethiosulfate, trypsin-digested and labeled using iTRAQ 8-plex reagents according to the manufacturer’s instructions (AB Sciex Inc., USA). A total of four independent iTRAQ experiments were conducted (Table S1, Supporting Information). The control replicates were labeled with iTRAQ tags 113, 114 and 115; the 200 mM NaCl replicates with tags 116 and 117; and the 400 mM NaCl replicates with tags 118, 119, and 121. Strong cation exchange (SCX) and reverse phase nanoflow LC−MS/MS were conducted as previously described.15 The iTRAQ MS/MS Data were processed by a thorough search considering biological modification and amino acid substitution against the NCBI green plant database using the Paragon algorithm26 of ProteinPilot software 4.0 (AB Sciex Inc., USA). Automatic decoy database searching were selected in both the Mascot and ProteinPilot software. Proteins were quantified on the basis of at least three confident MS/MS spectra (allowing generation of a p-value). A p-value smaller than 0.05 and fold change thresholds (1.2) were used. Four independent iTRAQ experiments were conducted using the leaf and root proteins from the M14 seedlings under different salt stress treatments.
RNA Sequencing and Data Analysis
Total RNAs from the control and treated samples were extracted using a Trizol reagent (Invitrogen, USA). RNA sequencing libraries were generated from the leaf and root samples and sequenced using Illumina HiSeq technology.27,28 Differential expressed genes (DEGs) were determined as previously described.27,28
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RESULTS AND DISCUSSION
Morphological Changes under Salt Stress
The M14 seedlings were grown in hydroponics with 0 (control), 200 mM and 400 mM NaCl. Morphological changes C
dx.doi.org/10.1021/pr400177m | J. Proteome Res. XXXX, XXX, XXX−XXX
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Figure 2. 2D-DIGE gels of root protein samples from plants grown in different salt stress conditions. (A) Control; (B) 200 mM NaCl; and (C) 400 mM NaCl. A total of 28 spots with significant volume changes under salt stress were labeled in (A) using the DeCyder software.
contained more than one protein (Table S5, Supporting Information). Therefore, we focused on the 86 protein identities representing 67 unique proteins in leaves and 22 protein identities representing 22 unique proteins in roots (Tables 1 and 2; Tables S2 and S3, Supporting Information). It was interesting to note that there were 10 proteins, and each was identified in two spots in leaf samples. They were RuBisCO large subunit-binding protein subunit alpha (spots 1957 and 1956), RuBisCO large subunit-binding protein subunit beta (spots 2135 and 2085), RuBisCO large subunit (spots 1172 and 1176), heat shock cognate 70 kDa protein (spots 1698 and 1688), chloroplastic peptidyl-prolyl cis−trans isomerase CYP20−3 (spots 5772 and 5961), 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (spots 1527 and 1515), vacuolar H+-ATPase subunit B (spots 2190 and 2194), pentatricopeptide repeat-containing protein At4g14850 (spots 1064 and 1007), 12S seed storage protein CRU1 (spots 1305 and 1508) and NBS-LRR disease-resistance protein scn3r1 (spots 6282 and 3453). In roots, two proteins, each identified in two spots, were heat shock cognate 70 kDa protein 4 (spots1050 and 1244) and heat shock cognate protein 80 (spots 972 and 954). In leaves, four proteins were found in multiple different spots. They were RuBisCO large subunit (spots 5025, 1449 and 1452), chloroplastic stromal 70 kDa heat shock-related protein (spots 1600, 1609 and 1611), carbonic anhydrase (spots 4080, 4159 and 3971), and methionine synthase (spots 1451, 1512, 1517 and 6940). Further inspection of the gel patterns revealed that the experimental mass and/or isoelectric point values of the spots differed from their theoretical values. This could be due to post-translational modifications and translation from alternatively spliced mRNAs.29−31
were observed one week after the salt treatment (Figure S1A, Supporting Information). Compared with the control plants, 200 mM and 400 mM NaCl treated M14 grew slowly and the fully expanded leaves appeared slightly chlorotic. Apparently, 400 mM NaCl treatment was more effective. Despite the growth phenotype, the M14 seedlings could survive the 7-day 400 mM NaCl treatment. The results were consistent with our previous result.21 Identification of Salt Stress-Responsive Proteins using 2D-DIGE and LC−MS
Proteins from leaves and roots from control and salt-treated M14 plants were separated on 2D-DIGE and imaged (Figure S1B,C, Supporting Information). The images of leaf gels and root gels were analyzed using DeCyder Differential Analysis Software (Figures 1 and 2). Only the protein spots that exhibited reproducible changes under salt treatment (average ratios >1.5 or
