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Nuclear Proteomics Reveals the Role of Protein Synthesis and Chromatin Structure in Root Tip of Soybean during the Initial Stage of Flooding Stress Xiaojian Yin, and Setsuko Komatsu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00330 • Publication Date (Web): 12 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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Journal of Proteome Research

Nuclear Proteomics Reveals the Role of Protein Synthesis and Chromatin Structure in Root Tip of Soybean during the Initial Stage of Flooding Stress

Xiaojian Yin 1,2 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.

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

Running title: Flooding responsive nuclear proteins of soybean

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

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ABSTRACT: To identify the upstream events controlling the regulation of floodingresponsive proteins in soybean, proteomic analysis of nuclear proteins in root tip was performed. Using nuclear fractions, which were highly enriched, a total of 365 nuclear proteins were changed in soybean root tip at initial stage of flooding stress. Four exon-junction complex-related proteins and NOP1/NOP56, which function in upstream of 60S pre-ribosome biogenesis, were decreased in flooded soybean. Furthermore, proteomic analysis of crude protein extract revealed that the protein translation was suppressed by continuous flooding stress. Seventeen chromatin structure-related nuclear proteins were decreased in response to flooding stress. Out of them, histone H3 was clearly decreased with protein abundance and mRNA expression levels at the initial flooding stress. Additionally, a number of protein synthesis-, RNA-, and DNArelated nuclear proteins were decreased in a time-dependent manner. mRNA expressions of genes encoding the significantly changed flooding-responsive nuclear proteins were inhibited by the transcriptional inhibitor, actinomycin D. These results suggest that protein translation is suppressed through inhibition of pre-ribosome biogenesis- and mRNA processing-related proteins in nuclei of soybean root tip at initial flooding stress. In addition, flooding stress may regulate histone variants with gene expression in root tip.

KEYWORDS: soybean, nuclei, flooding stress, root tips, pre-ribosome biogenesis, chromatin structure


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INTRODUCTION Nuclear proteins play a central role in the regulation of gene expression and protein synthesis. Due to the importance of nuclear proteins, nuclear proteome analyses have been performed in different plant species. For example, the use of this approach in Arabidopsis revealed that the plant responses to cold stress were controlled by multiple signaling pathways and cross-talk regulation 1. In tomato, nuclear proteomic analysis demonstrated that specific E2 ubiquitin-conjugating enzymes were involved in fruit ripening processing 2. The nuclear proteomic analysis of dehydration-sensitive and tolerant cultivars of chickpea identified a number of dehydration-responsive proteins that were potentially suitable molecular targets for increasing the adaptability of plants to water stress 3. In addition, a comparison among dehydration-responsive nuclear proteins of rice, legume, and chickpea revealed that although the dehydration response involved a few conserved proteins, most of the proteins were plant specific 4. Furthermore, several transcription factors, including Utp3 protein, were found to play an important role in rice seed germination through interacting with nuclear phosphoproteins 5. The findings from these nuclear proteomic studies have provided insight into plant biological processes such as ripening and germination, and the mechanisms by which plants adapt to abiotic stresses. Soybean is a flooding-sensitive crop that exhibits markedly reduced growth and grain yields in response to flooding stress 6. Proteomic analysis at the subcellular level to identify the mechanisms underlying flooding stress in soybean demonstrated that several cell wall proteins related to the reactive oxygen species scavenging system and jasmonate biosynthesis were decreased under flooding 7. Similar studies have also found that protein synthesis and glycosylation in the endoplasmic reticulum were affected by flooding stress 8. Proteomic analyses of mitochondria have indicated that 3 ACS Paragon Plus Environment


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proteins related to the tricarboxylic acid cycle are increased in soybean under flooding, whereas proteins comprising electron transport chain complexes were decreased 9. In addition, the receptor for activated protein kinase C1 was increased in abundance in flooding-stressed soybean 10, whereas poly-ADP-ribose polymerases were decreased 11. Examination of soybean nuclear proteomic profiles also revealed that abscisic acid enhanced the flooding tolerance of soybean through regulating zinc finger and cell division cycle 5 proteins 12. Nuclear phosphoproteomic analysis further indicated that abscisic acid affected the flooding response of early-stage soybean through the regulation of nuclear-localized phosphoproteins 13. Taken together, these findings suggest that different organ-specific proteins, particularly nuclear proteins, play important roles in regulating flooding stress-responsive pathways in soybean. Although it has been demonstrated that nuclear phosphoproteins played an important role in mediating tolerance to the initial stages of flooding stress in soybean 13

, the profile and interactions of nuclear proteins in early-stage soybean have not been

extensively studied. To identify novel flooding-responsive nuclear proteins in soybean, more sensitive nuclear proteomic analysis techniques, particularly for root tip proteins, are needed. In this study, to explore the upstream regulatory mechanisms controlling the expression and function of flooding-responsive proteins in the early stages of soybean growth, a gel-free proteomic analysis of enriched nuclei fraction was performed. Specifically, nuclear proteins were extracted from the root tip of flooding-stressed soybean using an improved nuclei enrichment method and peptides were then identified using a gel-free proteomic technique.

EXPERIMENTAL PROCEDURES Experimental Design and Statistical Rationale 4 ACS Paragon Plus Environment

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Two-day-old soybeans were treated without or with flooding stress for 3, 6, and 24 h (Figure 1). Nuclei were enriched from root tip and the purity of enriched nuclei fraction was analyzed using Western blot and enzyme activity assays. Nuclear proteins were extracted from enriched nuclei fraction and applied for gel-free proteomics. The function of the changed nuclear proteins was classified using MapMan bin codes. Furthermore, nuclear proteins were analyzed using bioinformatics such as Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) 14

. The results from bioinformatics were confirmed with mRNA expressions and protein

abundance such as Western blot and proteomics of crude protein extract. Three independent experiments were performed as biological replicates for all experiments. The statistical signiﬁcance of results was evaluated with the Student’s t-test when only two groups were compared. The statistical signiﬁcance of difference between multiple groups was evaluated using the one-way ANOVA test with Tukey’s multiple comparison. A p value of less than 0.05 was considered as statistical significance. All calculations were performed using SPSS software (version 12.0J; IBM Crop. Armonk, NY, USA).

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 of silica sand with 150 mL of 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-2sec-1, 16 h light period/day) at 25°C. For proteomic analysis, 2-day-old soybeans were treated without or with water for 3, 6, and 24 h. For mRNA expression analysis, 2-day-old soybeans were flooded without or with 5 µg/mL actinomycin D (Wako Pure Chemicals, Osaka, Japan) for 1.5, 3, and 6 h. After treatment, root tip was collected as sample. For initial flooding 5 ACS Paragon Plus Environment


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stress, samples from 3 time points containing 48 h, 51 h, and 51 h with 3-h-flooding were used. For continuous flooding stress, samples from 5 time points containing 48 h, 54 h, 54 h with 6-h-flooding, 72 h, and 72 h with 24-h-flooding were applied. Three independent experiments were performed as biological replicates for all experiments.

Nuclei Isolation and Protein Extraction Nuclei were isolated according to the manufacture’s instructions of Plant Nuclei Isolation/Extraction Kit (Sigma, St. Louis, MO, USA) with some modifications (Figure S1). Briefly, a portion (2.0 g) of samples was ground with buffer. The homogenates were filtered through a double layer of Filter Mesh and then centrifuged. The resulting pellet was resuspended in Nuclei Isolation buffer containing protease inhibitor mixture (Roche, Werk Penzberg, Germany), and then layered on top of cushions containing 60% percoll prepared in 1xNuclei Isolation buffer and 2.3 M sucrose. After centrifugation at 3,200 x g for 30 min at 4°C, the middle layer was collected and washed with Nuclei Isolation buffer containing protease inhibitor mixture to remove percoll and sucrose. The purified nucleus was vortexed with extraction buffer and sonicated. After sonication, the homogenate was centrifuged at 12,000 x g for 30 min at 4°C and the supernatant was collected as nuclear proteins.

Extraction of Crude Proteins A portion (0.5 g) of samples was ground to powder in liquid nitrogen using a mortar and pestle. Crude proteins were extracted as described by Komatsu et al. 12. The final pellet of crude protein extract was dried and resuspended by vortexing for 60 min at 25°C in lysis buffer consisting of 8 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine. The suspension was then centrifuged at 20,000xg for 20 min at 25°C 6 ACS Paragon Plus Environment

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and the supernatant was collected as crude protein extract.

Measurement of Protein Concentration For protein samples prepared for mass spectrometry (MS) analysis and enzyme activity analysis, the protein concentration was determined using the Bradford method 15

with bovine serum albumin as the standard. For protein samples prepared for Western

blot analysis, the protein concentration was measured with a Pierce 660 nm Protein Assay Kit with Ionic Detergent Compatibility Reagent (Thermo Fisher Scientific, Rockford, IL, USA).

Western Blot Analysis Proteins were extracted using SDS sample buffer containing 60 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol. Proteins were separated on a 12% SDS–polyacrylamide gel electrophoresis gel and then transferred onto a polyvinylidene difluoride membrane using a semidry transfer blotter. The blotted membrane was incubated overnight at 4°C in blocking buffer consisting of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% skim milk (Difco, Sparks, MD, USA). After blocking, the membrane was incubated with a 1:8,000 anti-histone H3 antibody (Abcam, Cambridge, UK) for 1 h at room temperature. Anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad, Hercules, CA, USA) was used as the secondary antibody. After a 1-h incubation with the secondary antibody, signals were detected using an ECL plus Western blotting detection kit (Nacalai Tesque, Kyoto, Japan) following the manufacturer’s protocol, and the signals were visualized using a LAS-3000 luminescent image analyzer (Fujifilm, Tokyo, Japan). Coomassie brilliant blue staining was used as loading control. The relative intensities of bands were 7 ACS Paragon Plus Environment


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calculated using Quantify One software (version 4.5; Bio-Rad).

Enzyme Activity Analysis Proteins were extracted using buffer containing 50 mM HEPES-NaOH (pH 7.5), 5 mM MgCl2, 1mM EDTA, 2% polyvinylpyrrolidone, 0.1% Triton X-100, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride. For alcohol dehydrogenase assay, the reaction solution was composed of 50 mM MES-NaOH, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM NADH, and 4% acetaldehyde. NADH oxidation was monitored at 340 nm at 25°C for 5 min. The enzyme activity was calculated with formula: µnits/mL = (∆A340 x total volume x dilution factor) / (6.22 x sample volume) 16

. For catalase activity, the reaction buffer was composed of 50 mM potassium

phosphate and 15 mM H2O2. The subsequent decomposition of H2O2 was measured at 240 nm at 25°C for 5 min. The enzyme activity was calculated with formula: µnits/mL = (∆A240 x total volume x dilution factor) / (40 x sample volume) 17. For fumarase activity, the reaction buffer was consisted of 70 mM KH2PO4-NaOH, 0.05 % Triton X-100, and 50 mM malic acid. The reaction was directly measured at 340 nm. The enzyme activity was calculated with formula: µnits/mL = (∆A340 x total volume x dilution factor) / (2.55 x sample volume) 18. For NADH-cytochrome c reductase activity, the reaction buffer contained 20 mM potassium phosphate (pH 7.2), 0.2 mM NADH, 0.02 mM cytochrome c, and 30 mM NaN3. The reduction of cytochrome c was followed spectrophotometrically as the absorbance increase at 550 nm. The enzyme activity was calculated with formula: µnits/mL = (∆A550 x total volume x dilution factor) / (21.1 x sample volume) 19.


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Enrichment and Digestion for Nuclear Proteins and Crude Proteins Proteins (200 µg) were enriched by phase separation in the organic layer. The enriched proteins were reduced, alkylated, and digested as described by Yin et al. 20. The resulting tryptic peptides were acidified with formic acid (pH < 3), and the mixed solution was centrifuged at 20,000 x g for 10 min. The supernatant was collected for liquid chromatography (LC)-MS/MS analysis.

Mass Spectrometry Analysis Peptides in 0.1% formic acid were loaded onto an Ultimate 3,000 nanoLC 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, nanoLC capillary column; Nikkyo Technos, Tokyo, Japan) with a spray voltage of 1.8 kV. The peptide ions in the spray were analyzed on a nanospray LTQ XL Orbitrap MS (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 MS over 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy 21. As the lock mass, the ions C24H39O4+ (m/z 391.28429), C14H46NO7Si7+ (m/z 536.16536), and C16H52NO8Si8+ (m/z 610.18416) were used. The 3 most intense precursor ions above a threshold of 500 were selected for collision-induced fragmentation in the linear ion trap at a normalized collision energy of 35% after accumulation to a target value of 1,000. Dynamic exclusion was employed within 90 sec to prevent the repetitive selection of peptides. Acquired spectra were used for protein identification. 9 ACS Paragon Plus Environment


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Protein Identification using Acquired Mass Spectrometry Data Protein identification was performed using the Mascot search engine (version 2.5.1, Matrix Science, London, UK) and a soybean peptide database (54,175 sequences) obtained from the soybean genome database (Phytozome version 9.1, http://www.phytozome.net/soybean) 22. DTA files were generated from acquired raw data files and then converted to Mascot generic files using Proteome Discoverer software (version 1.4.0.288, Thermo Fisher Scientific). 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. Trypsin was specified as the proteolytic enzyme, and one missed cleavage was allowed. Peptide mass precursor tolerance was set at 10 ppm, fragment mass tolerance was set at 0.8 Da, and peptide charges were set at +2, +3, and +4. Peptide cut-off score was 10 and for peak filtration the S/N threshold (FT-only) was set at 1.5. 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 23

. False discovery rates (false positive/ (false positive+true postive) ) for peptide

identification of all searches were less than 1.0%. Peptides with a percolator ion score of more than 13 (p

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