Phosphoproteomics Reveals the Effect of Ethylene in Soybean Root

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Phosphoproteomics Reveals the Effect of Ethylene in Soybean Root under Flooding Stress Xiaojian Yin, Katsumi Sakata, and Setsuko Komatsu J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 14 Oct 2014 Downloaded from http://pubs.acs.org on October 16, 2014

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Journal of Proteome Research

Phosphoproteomics Reveals the Effect of Ethylene in Soybean Root under Flooding Stress

Xiaojian Yin 1,2, Katsumi Sakata3, and Setsuko Komatsu 1,2, *

1

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba

305-8572, Japan. 2

National Institute of Crop Science, National Agriculture and Food Research

Organization, Tsukuba 305-8518, Japan. 3

Maebashi Institute of Technology, Maebashi 371-0816, Japan.

* Corresponding Author: Setsuko Komatsu, National Institute of Crop Science, National Agriculture and Food Research Organization, Kannondai 2-1-18, Tsukuba 305-8518, Japan. Tel: +81-29-838-8693, Fax: +81-29-838-8694, Email: [email protected]

Running title: Phosphoproteomics of Soybean under Flooding Stress

Abbreviations: LC, liquid chromatography; MS, mass spectrometry; qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

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ACS Paragon Plus Environment


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ABSTRACT: Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth, but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4G was located in the center of network during flooding. Soybean eukaryotic translation initiation factor 4G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment of an ethylenereleasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role via the protein phosphorylation in plant tolerance mechanisms to the initial stages of flooding stress in soybean root tips.

KEYWORDS: soybean, flooding stress, root tip, phosphoproteomics 2


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INTRODUCTION With the continuing trend of global warming, the frequency of heavy rains and flooding is expected to rise 1. Flooding, which is a widespread environmental stress, has severe negative effects on the productivity of arable farmland, as the majority of crops cannot grow under flooding conditions 2. Flooding stress reduced gas exchange between plant and atmosphere, because oxygen diffuses more than 10,000 times slowly in water than in air 3. The resulting decrease in oxygen concentration leads to impaired mitochondrial oxidative phosphorylation in plant cells and a corresponding decrease in ATP 4. To produce sufficient ATP to support survival in low oxygen conditions, plants initially respond by activating anaerobic pathways to generate ATP through glycolysis and ethanol fermentation metabolism 5. Soybean is a flooding-intolerant crop whose growth and grain yields are markedly reduced by flooding stress 6. Despite intensive study of this agriculturally important crop, the mechanisms underlying against flooding stress in soybean are not well characterized. Injury of soybean seeds before radical protrusion was caused by the physical disruption of cells under flooding stress due to the rapid uptake of water and can be alleviated or minimized by sowing seeds with a high moisture content 7. Hashiguchi et al. 8 reported that the elongation of soybean roots under flooding stress was suppressed in the first 24 h and was significantly retarded after 48 h. Analysis of soybean gene expression in the first 12 h of flooding stress has shown that genes associated with alcohol fermentation, ethylene biosynthesis, pathogen defense, and cell loosening are significantly up-regulated 9. Taken together, these findings indicate that plant responses in the early stages of flooding are the most critical for soybean growth and survival. Reversible modification of plant proteins by phosphorylation is a common 3



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signaling event that occurs in response to both abiotic and biotic stresses. In the case of maize, sucrose synthase 10 and eukaryotic initiation factor 4E 11 are phosphorylated, whereas ribosomal proteins 12 are dephosphorylated in oxygen-deprived roots. In soybean, a gel-based phosphoproteomic analysis revealed that proteins related to protein folding and synthesis were dephosphorylated under flooding stress 13. Furthermore, mass spectrometry (MS)-based phosphoprotein analysis showed that soybean responses to flooding stress were regulated by both changes in protein abundance and phosphorylation status, and that energy-demanding and production-related metabolic pathways were particularly sensitive to changes in protein phosphorylation during flooding 14. The results from these studies indicate that protein phosphorylation plays a significant role in modulating the adaptive responses of soybean under flooding conditions. However, the relationships and interactions between flooding-responsive phosphoproteins proteins have not been well characterized in soybean. To explore the flooding-responsive mechanisms in early-stage soybean, here, a phosphoproteomic approach was used to identify and characterize phosphorylated proteins in the early stages of flooding stress. Phosphopeptides from root tips were enriched using a polymer-based metal-ion affinity capture technique and identified using a gel-free proteomic technique. In addition, to identify the key phosphoproteins involved in the soybean flooding response, protein-protein interactions between differentially expressed phosphoproteins were analyzed using hierarchical clustering based on S-system differential equations. Furthermore, gene expression levels, and protein and phosphoprotein abundance were compared between control and flooding-stress conditions.

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EXPERIMENTAL PROCEDURES Plant Material and Treatments Seeds of soybean (Glycine max L. cultivar Enrei) were sterilized with 1% sodium hypochlorite solution, rinsed in water, and sown on 500 mL silica sand moistened with 125 mL water in a plastic case (180 mm x 140 mm x 45 mm). Soybeans were grown in a growth chamber illuminated with white fluorescent light (160 µmol m-2s-1, 16 h light period/day) at 25 °C and 70% relative humidity. Two-day-old soybeans were treated without (control) or with flooding for 3, 6, 12, and 24 h. Additionally, two-day-old soybeans were transferred to column tubes containing 120 mL water, 300 ppm ethephon, and 5 mM silver nitrate under darkness at room temperature for 0, 3, 6, and 12 h. At the end of the respective treatment periods, 5 mm-length root tips were collected and immediately frozen in liquid nitrogen. Three independent experiments were performed as biological replicates.

Measurement of ATP Content A portion (250 mg) of each collected soybean root tip sample was ground in liquid nitrogen with a mortar and pestle and was then mixed with 1 mL ATP extraction buffer, which consisted of 0.51 M trichloroacetic acid and 17 mM EDTA. After sonication for 10 min, the sample was centrifuged at 20,000 x g for 5 min, and the resulting supernatant was collected for measurement of the ATP content. For the reaction, 100 µL of the supernatant sample was added to 900 µL reaction solution, consisting of 280 mM HEPES-NaOH, 4 mM MgCl2, 2 mM glucose, 2 mM NAD, and 4 U/mL glucose-6-phosphate dehydrogenase, and the absorbance at 340 nm was then immediately measured using a spectrophotometer (DU730, Beckman, Indianapolis, IN, USA). Hexokinase (4 U/mL) was added to the reaction solution, which was then 5



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incubated at 25 °C for 30 min, and the absorbance at 340 nm was again measured. ATP content was determined from the increase in absorbance at 340 nm before and after the addition of hexokinase.

Protein Extraction and Digestion for Proteomic Analysis for Total Proteins A portion (500 mg) of each collected root tip sample was ground in liquid nitrogen with a mortar and pestle and then mixed with extraction buffer composed of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, phosphatase inhibitor mixture (Sigma, St. Louis, MO, USA), and protease inhibitor mixture (Roche, Werk Penzberg, Germany). After centrifugation at 12,000 x g for 20 min, the obtained supernatant was diluted with three volumes cold acetone containing 0.07% 2-mercaptoethanol and then incubated at -20 °C for 2 h. The resulting precipitate was collected and washed three times with an acetone solution containing 0.07% 2-mercaptoethanol. Finally, the protein pellets were dried in a Speed-Vac and resuspended in lysis buffer which consisted of 7 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine. The protein concentration was then measured by the Bradford method 15 with bovine serum albumin as the standard. The extracted root tip proteins (150 µg) were purified as follows. After adjusting the volume of the dried protein samples to 150 µL, 600 µL of methanol was added and the resulting solution was thoroughly mixed. Subsequently, 150 µL of chloroform was added to the solution, which was again mixed. To induce phase separation, 450 µL water was added to the solution, and the resulting mixture was vortexed and then centrifuged at 20,000 x g for 5 min. The upper aqueous phase was discarded, and 450 µL methanol was added to the organic phase. The samples were centrifuged at 20,000 x g for 5 min, the resulting supernatants were discarded, and the pellets were dried. 6


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The dried pellets were resuspended in 50 mM NH4HCO3, and proteins in the samples were then reduced, alkylated, and digested using trypsin and lysyl endopeptidase at a 1:100 enzyme/protein concentration at 37 °C for 16 h. The treated peptides were desalted using a MonoSpin C18 column (GL Sciences, Tokyo, Japan).

Protein Extraction and Digestion for Phosphoproteomic Analysis A portion (500 mg) of each collected root tip sample was ground in liquid nitrogen with a mortar and pestle and then mixed with extraction buffer, containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, phosphatase inhibitor mixture, and protease inhibitor mixture. After centrifugation at 12,000 x g for 20 min, the obtained supernatant was diluted with three volumes cold acetone containing 0.07% 2-mercaptoethanol and then incubated at −20 °C for 2 h. The resulting precipitate was collected and washed three times with an acetone solution containing 0.07% 2-mercaptoethanol. Finally, the protein pellets were dried in a Speed-Vac and resuspended in lysis buffer. After measuring the protein concentration and purifying the samples by the same method used for the proteomic analysis for total proteins, the samples were reduced, alkylated, and digested using trypsin at a 1:100 enzyme/protein concentration at 37 °C for 16 h. The resulting peptides were desalted with a MonoSpin C18 column.

Phosphopeptide Enrichment Polymer-based Metal-ion Affinity Capture Phosphopeptide Enrichment Reagent (Tymora, West Lafayette, IN, USA) was used to enrich phosphopeptides. Desalted peptides (150 µg) were resuspended in 100 µL loading buffer, consisting of 100 mM glycolic acid, 1% trifluoroacetic acid, and 50% acetonitrile, to which 10 µL 7



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polymer-based metal ion affinity capture-Ti reagent was added. After shaking the resulting mixture for 30 min, 200 µL of 300 mM HEPES (pH 7.7) was added to increase the pH of the sample above 6.3. After brief vortexing, 50 µL magnetic capture beads were added to the sample, which was then shaken vigorously for 10 min. The beads were then separated from the solution using a magnetic separator rack (Invitrogen Dynal AS, Oslo, Norway). Captured beads were washed once in loading buffer and twice in washing buffer, consisting of 100 mM acetic acid, 1% trifluoroacetic acid, and 80% acetonitrile. Phosphopeptides were then eluted by washing the beads twice with 100 µL of 400 mM NH4HCO3.

Nanoliquid Chromatography-Tandem Mass Spectrometry Analysis Peptides in 0.1% formic acid were loaded onto an Ultimate 3,000 Nano-liquid Chromatography (LC) system (Dionex, Germering, Germany) equipped with a C18 PepMap trap column (300 µm ID × 5 mm, Dionex). The peptides were eluted from the trap column and were then separated using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 µm 1D × 120 mm, NTTC-360/75-3 nano-LC capillary column; Nikkyo Technos, Tokyo, Japan) with a spray voltage of 1.8 kV. The peptide ions in the spray were detected in the Peptides were analyzed on a nanospray LTQ XL Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Full-scan mass spectra were acquired in the Orbitrap MS over 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy 16. As the lock mass, the ions C24H39O4+ (m/z 391.28429), C14H46NO7Si7+ (m/z 536.16536), and C16H52NO8Si8+ (m/z 610.18416) were used. Values for ion isolation window were set as follows: activation 8


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type was collision-induced dissociation (CID), default charge state was 2, isolation width was 2.0 m/z, normalized collision energy was 35.0, and activation time was 30.000 ms. Values for dynamic exclusion were determined as follows: repeat count was 2, repeat duration was 30.0 sec, exclusion list size was 500, exclusion duration was 90.0 sec, and exclusion mass width was ± 1.5 Da. The 3 most intense precussor ions above a threshold value of 500 were selected for collision-induced fragmentation. The acquired MS spectra were used for protein identification.

Protein Identification Protein identification was performed using the MASCOT search engine (version 2.3.0.2, Matrix Science, London, UK) and a soybean peptide database (55,787 sequences) obtained from the soybean genome database (Phytozome version 8.0, http://www.phytozome.net/soybean) 17. DTA files were generated from acquired raw data files and then converted to MASCOT generic files using Protein Discover software (version 3.3.1, Thermo Fisher Science). The parameters used in the MASCOT searches were as follows: carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. For peptides after phosphopeptide enrichment, carbamidomethylation of cysteine was set as a fixed modification, and phosphoST, phosphoY, and oxidation of methionine were set as variable modifications. For both normal peptides and peptides after phosphopeptide enrichment, trypsin was specified as the proteolytic enzyme, and one missed cleavage was allowed. Peptide mass tolerance was set at 5 ppm, fragment mass tolerance was set at 0.5 Da, and peptide charges were set at +2, +3, and +4. An automatic decoy database search was performed as part of the search. MASCOT results were filtered with the MASCOT percolator to improve the accuracy and sensitivity of peptide identification 18. 9



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False discovery rates for peptide identification of all searches were less than 1.0%. Peptides with a percolator ion score of more than 13 (p 0.9 were considered as candidate interactions and were further evaluated as model interactions.

RNA Extraction and Quantitative Reverse Transcription-Polymerase Chain Reaction Analysis 11



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A portion (100 mg) of each root tip sample was ground into powder in liquid nitrogen using a sterilized mortar and pestle. Total RNA was extracted from the homogenized tissue using an RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA) and was then reverse-transcribed using an iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer’s instructions. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed in a 10-µL reaction mixture using SsoAdvanced SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and a MyiQ Single-Color Real-Time PCR Detection system (Bio-Rad). The PCR conditions were as follows: 95°C for 30 sec, followed by 45 cycles of 95°C for 10 sec and 60°C for 30 sec. Gene expression was normalized using 18S rRNA as an internal control. The primers were designed using the Primer3 web interface (http://frodo.wi.mit.edu) and are listed in Supplemental Table 1. Primer specificity was checked by BLASTN searches against sequences in the soybean genome database (Phytozome) with the designed primers as queries by melt curve analysis. Amplified fragments were detected using agarose gel electrophoresis.

Statistical Analysis The statistical significance of the results was evaluated by the Student’s t-test when only two groups were compared. The statistical significance of comparisons between multiple groups was evaluated with One-way ANOVA test. A p value of

Phosphoproteomics Reveals the Effect of Ethylene in Soybean Root

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