Proteomic Analysis of Phosphoproteins in the Rice Nucleus During the

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Proteomic Analysis of Phosphoproteins in the Rice Nucleus during Early Stage of Seed Germination Ming Li, Xiaojian Yin, Katsumi Sakata, Pingfang Yang, and Setsuko Komatsu J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 02 Jun 2015 Downloaded from http://pubs.acs.org on June 3, 2015

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Journal of Proteome Research

Proteomic Analysis of Phosphoproteins in the Rice Nucleus during Early Stage of Seed Germination

Ming Li1, 2,#, Xiaojian Yin2,#, Katsumi Sakata3, Pingfang Yang1, and Setsuko Komatsu2,*

1 Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China 2 National Institute of Crop Science, National Agriculture and Food Research Organization, Tsukuba 305-8518, Japan 3 Department of Life Science and Informatics, Maebashi Institute of Technology, Maebashi 371-0816, Japan

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

Running title: Nuclear phosphoproteomics during rice germination

ABBREVIATIONS ABA, abscisic acid; GAs, gibberellins; LC, liquid chromatography; MS, mass spectrometry; PolyMAC, Polymer-based Metal-ion Affinity Capture; qRT-PCR, quantitative reverse transcription polymerase chain reaction

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ABSTRACT: The early stage of seed germination is the first step in the plant life cycle without visible morphological change. To investigate the mechanism controlling the early stage of rice seed germination, a gel-free/label-free nuclear phosphoproteomics was performed. A total of 3,467 phosphopeptides belonging to 102 nuclear phosphoproteins of rice embryos were identified. Protein synthesis related proteins were mainly phosphorylated. During the first 24 h following imbibition, 115 nuclear phosphoproteins were identified and significant changes in phosphorylation level over time were observed in 29 phosphoproteins. Cluster analysis indicated that nucleotide binding proteins and zinc finger CCCH/BED type proteins increased in abundance during the first 12 h of imbibition and then decreased. The in silico protein-protein interactions for 29 nuclear phosphoproteins indicated that Sas10/Utp3 protein, which functions in snoRNA bind and gene silence, was the center of the phosphoproteins network in nuclei. The germination rate of seeds was significantly slow down with phosphatase-inhibitor treatment. The mRNA expression of zinc finger CCCH type protein did not change and zinc finger BED type was up-regulated in rice embryos during early stage of germination with phosphatase-inhibitor treatment. These results suggest that phosphorylation and dephosphorylation of nuclear protein involve in rice seed germination. Furthermore, transcription factors such as zinc finger CCCH/BED type protein might play a key role through nuclear phosphoproteins, and Sas10/Utp3 protein might be interacted with nuclear phosphoproteins in rice embryos to mediate the early stage of seed germination.

KEYWORDS: rice, nucleus, phosphoproteomics, germination, phosphonetwork


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INTRODUCTION For many species of plants, seeds are necessary to reproduce and spread offspring, and seed production is also a good strategy to overcome severe environmental conditions 1. Seed germination, which usually starts when water is imbibed by the quiescent dry seed and ends with the elongation of the radicle, is a key stage in the plant life cycle 2,3. Based on the increase in seed weight, rice seed germination is divided into three phases: first, rapid water uptake without visible morphological change (Phase I), followed by a plateau stage (Phase II) without any apparent change in weight, and finally, rapid water uptake (Phase III) accompanied by growth of the radical and coleoptile 3. Transcriptomic analysis of rice indicated that the switch involved in germination may happen in Phase I, with the onset of mRNA biosynthesis 4. Among three phases, Phase I is the important stage during seed germination because mRNA biosynthesis starts at this stage. To understand the mechanism of rice seed germination, a proteomic technique was used. Exploring gibberellins (GAs) and abscisic acid (ABA) responsive functional proteins such as DELLA in the GA and ABA signaling pathways suggested many proteins are involved in GA and ABA modulation during seed germination 5,6. Profiling of rice seed protein abundance during germination revealed comprehensive metabolic and regulatory pathways during seed germination 7. Study of embryos revealed that starch biosynthetic enzymes play major roles in rice seed germination 8. Investigation of coleoptiles indicated that they respond to anoxia dramatically, with an increase in abundance of proteins related to stress responses and redox metabolism 9-11. Analysis of the aleuronic layer has demonstrated that thioredoxin activates embryo-specific protein 2 with a concurrent unfolding of its substrate during rice seed germination 12-14. Because the function of a protein is largely correlated with its cellular location and posttranslational modifications, research on subcellular proteomics and posttranslational 3 ACS Paragon Plus Environment


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modifications might be helpful in understanding the mechanism controlling rice seed germination. In order to clarify this mechanism, mitochondrial proteomics and phosphoproteomics studies have been performed during germination of rice seeds. In rice seed mitochondrial proteomics, it was found that mitochondria matured during germination in anaerobic conditions, and respiratory functionality was repressed by reduced protein abundances of tricarboxylic acid cycle components and cytochrome-containing complexes of the respiratory chain 15,16. In rice seed phosphoproteomics, it was found that brassinosteroid signaling might participate in the triggering of rice seed germination 17,18. However, proteomic studies of other organelles or posttranslational modifications are still lacking. The nucleus is responsible for storing most genetic information and controlling the plant's activities 19. All nuclear proteins are synthesized in the cytoplasm and imported into the nucleus to execute their functions 20,21. Furthermore, after the plasma membrane perceives changes in the extracellular environment, signals must be communicated to the nucleus to induce or suppress gene transcription 21. Phosphorylation plays a key role in the signalling cascade through kinases and phosphatases to activate or inactivate enzyme activity in biological pathways 22. As a reversible protein modification, phosphorylation enhances or inhibits proteins transport between nucleus and cytoplasm 23. Some studies on regulation and activation of nuclear localized proteins during seed germination have focused on a few individual proteins using genetic techniques 24-26. However, it is difficult to carry out large-scale identification of phosphorylation sites in nuclear proteins during rice seed germination. Phosphorylation plays a key role in protein transport between nucleus and cytoplasm 23. To understand the role of nuclear proteins and their phosphorylation during germination of rice seeds, a gel-free/label-free proteomic technique was used. After extraction and 4 ACS Paragon Plus Environment

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digestion of nuclear proteins, phosphopeptides were enriched and analyzed using a phosphopeptide enrichment reagent. These nuclear phosphopeptides were analyzed using nano liquid chromatography (LC) coupled tandem mass spectrometry (MS). In addition, to identify the key nuclear phosphoproteins involved in rice seed germination, in silico protein-protein interactions were analyzed using hierarchical clustering and the S-system differential equation. Furthermore, gene expression levels of key nuclear phosphoproteins were analyzed using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and were compared with the abundance of phosphoproteins.

EXPERIMENTAL PROCEDURES Plant Material, Seed Germination, and Treatment Seeds of rice (Oryza sativa cultivar Nipponbare) were dehusked and placed in culture plates (10 cm x 10 cm) for germination. Rice seeds were germinated on moist mud bed in agriculture and wet filter paper in this experiment 7,27. Seeds were imbibed for 12 h or 24 h in deionized water in a dark growth chamber at 25ºC. Embryos were separated from seeds after 0, 12, or 24 h of imbibition using a knife blade and collected on ice. In total, 50 seeds were prepared for proteomic experiments and 1,400 for nuclear phosphoproteomics experiments. For RNA experiments, 50 seeds were prepared. Rice seeds were treated with 0.01% dimethyl sulfoxide (control), 1% phosphatase inhibitor (Sigma, St. Louis, MO, USA), 10 µM GAs (Wako, Osaka, Japan), and 10 µM ABA (Tokyo Chemical Industry, Tokyo, Japan) solutions, and embryos were collected after 0, 12 and 24 h of treatments. Three independent experiments were performed as biological replicates for all experiments (Supplemental Figure 1).

Isolation of Nuclei and Extraction of Nuclear Proteins 5 ACS Paragon Plus Environment


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Nuclei were isolated using a Plant Nuclei Isolation/Extraction Kit (Sigma) following the manufacturer’s protocol with some modifications. Rice embryos (2 g) were ground with 5 mL 1x Nuclei Isolation buffer (Sigma) using a mortar and pestle. The homogenate was filtered through a double layer of Filter Mesh 100 (Sigma) and transferred to a 15 mL tube, then centrifuged at 1,300 x g for 10 min at 4°C. The resulting pellet was resuspended in Nuclei Isolation buffer containing protease inhibitor mixture (Roche, Werk Penzberg, Germany) and phosphatase-inhibitor mixture, then layered on top of a 60% Percoll/2.3 M sucrose cushion. Percoll was diluted to 60% using Nuclei Isolation buffer, and 2.3 M sucrose was supplied in kit. After centrifugation at 3,200 x g for 30 min at 4°C, the middle layer between 60% Percoll and 2.3 M sucrose was carefully collected with a Pasteur pipette. The purified nuclei were washed 2 times using Nuclei Isolation buffer. Nuclei were vortexed at 4°C for 20 min with extraction buffer (Sigma) containing protease inhibitor mixture and phosphatase-inhibitor mixture, and then sonicated in ice water for 20 min. Vortexing and sonication were repeated once. After sonication, the homogenate was centrifuged at 12,000 x g for 30 min at 4°C and the supernatant was collected as nuclear protein (Supplemental Figure 2). Protein concentrations were measured using the Bradford assay with bovine serum albumin as the standard 28.

Extraction of Total Proteins Rice embryos (0.1 g) were ground to powder in liquid nitrogen using a mortar and pestle. The powder was transferred to an acetone solution containing 10% trichloroacetic acid and 0.07% 2-mercaptoethanol, and the resulting mixture was vortexed and then sonicated for 10 min. The suspension was incubated for 1 h at -20°C with vortexing every 15 min and then centrifuged at 9,000 × g at 4°C for 20 min. The supernatant was discarded and the pellet was washed twice with 0.07% 2-mercaptoethanol in acetone. The pellet was dried using a 6 ACS Paragon Plus Environment

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Speed-Vac concentrator (Savant Instruments, Hickville, NY, USA) and then resuspended in lysis buffer consisting of 7 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine by vortexing for 1 h at 25°C. The suspension was centrifuged at 20,000 × g for 20 min at 25°C and the supernatant was collected as total protein. Protein concentrations were measured using the Bradford assay as above.

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 29. The protein concentration was measured with a Pierce 660 nm Protein Assay Kit with Ionic Detergent Compatibility Reagent (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were separated by electrophoresis on a 12% SDS-polyacrylamide 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 dilution of anti-histone H3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with a 1:3,000 dilution of anti-ascorbate peroxidase antibody (provided by Proteomic Laboratory in National Institute of Crop Science, Tsukuba, Japan) 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 Chemi-Lumi One Super 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 a loading control. The relative intensities of bands were calculated using Quantity One software (version 4.5, Bio-Rad). 7 ACS Paragon Plus Environment


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Purification and Digestion of Proteins Proteins (100 µg) were purified by phase separation in the organic layer. After adjusted the volume to 150 µL (100 µg), 600 µL methanol was added and the resulting solution was thoroughly mixed. Subsequently, 150 µL 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 and resuspended in 50 mM NH4HCO3. Proteins were reduced with 50 mM dithiothreitol for 30 min at 56°C, alkylated with 50 mM iodoacetamide for 30 min at 37°C in darkness, and digested with trypsin 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).

Phosphopeptide Enrichment Polymer-based Metal-ion Affinity Capture (PolyMAC) Phosphopeptide Enrichment Reagent (Tymora, West Lafayette, IN, USA) was used to enrich phosphopeptides. Desalted peptides (200 µg) were resuspended in 100 µL loading buffer consisting of 100 mM glycolic acid, 1% trifluoroacetic acid, and 50% acetonitrile, to which 10 µL PolyMAC-Ti reagent was added. After shaking the resulting mixture for 30 min, 200 µL 300 mM HEPES-NaOH (pH 7.7) was added to increase the pH of the sample above 6.3. After brief vortexing, 50 µL of magnetic capture beads were added to the sample, which was shaken vigorously for 10 min. The beads were 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 8 ACS Paragon Plus Environment

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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 400 mM NH4HCO3. After drying the eluent, the pellet was resuspended well in 10 µL 0.25% formic acid. The phosphopeptide solution was collected after centrifugation.

Mass Spectrometric Analysis Peptides in 0.1% formic acid were loaded onto an Ultimate 3,000 nano-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 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, 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 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 mass spectrometer over 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy 30. 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 type was collision-induced dissociation, 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 sec, exclusion list size was 500, exclusion duration was 90 sec, and exclusion mass width was ± 1.5 Da. The three most intense precursor ions above a threshold value of 500 were selected for collision-induced fragmentation. The acquired mass 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 rice protein database (50,253 sequences and 15,266,515 residues) obtained from The Rice Annotation Project Database (RAP-DB, http://rapdb.dna.affrc.go.jp), including protein sequences supported by FL-cDNA and EST data (IRGSP-1.0_protein_2013-4-24) and protein sequence predicted computationally (IRGSP-1.0_predicted protein_2013-3-9) 31,32. DTA files were generated from acquired raw data files and 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: 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 10 ppm, fragment mass tolerance was set at 0.8 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 33. False discovery rates for peptide identification of all searches were less than 1.0%. Peptides with a percolator ion score of more than 13 (p0.9 were considered candidate interactions. In the model protein interaction diagram, a red arrow indicates an inductive interaction and corresponds to gij > 0 in the S-system differential equation, and a blue T-bar indicates a suppressive interaction and corresponds to gij < 0 in the S-system differential equation 39.

RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction Analysis Rice embryos (0.1 g) were ground into fine powder in liquid nitrogen with a sterilized mortar and pestle. Total RNA was extracted from the powder using an RNeasy Plant Mini kit (Qiagen, Valencia, CA, USA). RNA was reverse-transcribed using an iScript cDNA synthesis kit (Bio-Rad) according to the manufacturer’s instructions. qRT-PCR was performed in a 10 µL reaction using SsoAdvanced SYBR Green Supermix (Bio-Rad) 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) (Supplemental Table 1). Specificity of primers 12 ACS Paragon Plus Environment

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was checked by BLASTN search against the RAP-DB with the designed primers as queries, by melt curve analysis and by agarose gel electrophoresis of amplified fragments.

Statistical Analysis The statistical significance of results was evaluated using Student’s t-test when only two groups were compared. The statistical significance of results from multiple groups was evaluated using a one-way ANOVA test. Both calculations were performed using GraphPad software (version 5.0, GraphPad Software, San Diego, CA, USA). A p value < 0.05 was considered to be statistically significant.

RESULTS Purity of Nuclear Proteins Isolated from Rice Embryos To understand the role of nuclear proteins and their phosphorylation during rice seed germination, nuclear proteins were isolated. Nuclei were isolated from rice embryos collected after 24 h of imbibition (Supplemental Figure 2). In total, 100 µg of nuclear proteins was extracted from 1 g of rice embryos. Western blotting with an anti-histone H3 and ascorbate peroxidase antibodies was performed to check the purity of nuclear proteins. The antiascorbate peroxidase antibody cross-reacted with 31-kDa proteins and the anti-histone H3 antibody cross-reacted with 17-kDa nuclear proteins (Figure 1). The relative intensity of cytosolic ascorbate peroxidase was markedly stronger in the total protein fraction than in the nuclear protein fraction. The strong intensity of bands corresponding to histone H3 indicated that the nuclei were significantly enriched. Total protein and nuclear proteins extracted from rice embryos were analyzed using nanoLC-MS/MS. A total of 349 total proteins (Supplemental Table 2) and 362 nuclear proteins (Supplemental Table 3) were identified. Comparing total proteins with nuclear 13 ACS Paragon Plus Environment


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proteins, 149 proteins were in common, 200 were specific to total proteins, and 213 were apparently specific to nuclear proteins (Figure 2A). In order to evaluate the purity of nuclear protein, the localization of each protein from the total protein and nuclear protein fractions was predicted using intracellular targeting prediction programs (Figure 2B); 71 (20%) of the proteins in the total protein and 196 (54%) of the nuclear proteins were predicted to be located in nuclei (Figure 2B). Proteins contaminating the nuclear fraction mainly belonged to the cytoplasm (16%) and chloroplast (18%) (Figure 2B). These results indicated that nuclear proteins in the nucleus fraction were enriched more than 2.5 times compared to total protein by the procedure.

Function of Nuclear Proteins in Rice Embryos after 24 h of Imbibition In order to understand functions of 362 nuclear proteins, they were categorized using MapMan bin codes. In total, 362 proteins were classified into 21 functional categories (Figure 3). Categories related to protein (39%), RNA (13%), development (6%), and DNA (6%) represented the largest number of proteins. The functional category of protein contained 141 proteins and this category was further classified into 4 groups, which were protein synthesis (83%), protein degradation (9%), protein targeting (6%), and others (2%) (Figure 3). Moreover, 47 proteins and 22 proteins respectively belonged to the RNA and DNA categories, with 16 nucleotide-, RNA-, and DNA-binding proteins belonging to the RNA category, and 8 histones belonging to the DNA category (Supplemental Table 3).

Phosphoproteomic Analysis of Nuclear Proteins in Rice Embryos after 24 h of Imbibition To understand the phosphorylation status of nuclear proteins, a phosphoproteomics study was performed. Nuclear proteins were extracted from rice embryos after 24 h of imbibition. Nuclear phosphopeptides were enriched and analyzed by nanoLC-MS/MS. 14 ACS Paragon Plus Environment

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Based on this technique, 3,467 phosphopeptides (Supplemental Table 4) belonging to 102 phosphorylated proteins (Supplemental Table 5) were identified. Overall, 65% and 30% of phosphorylated peptides respectively contained a single and double phosphoryl group (Figure 4A). Serine, threonine, and tyrosine phosphorylated peptides accounted for 64, 31, and 5%, respectively (Figure 4B). Nuclear phosphoproteins were classified into 14 functional categories (Figure 4C). Among these categories, protein, RNA, DNA, and cell contained the largest number of proteins, comprising 25, 21, 10, and 5%, respectively. The functional category of protein contained 25 proteins, and this category was further classified into 4 groups, which were protein synthesis (64%), protein degradation (24%), protein targeting (8%), and protein folding (4%) (Figure 4C).

Changes in Abundance of Nuclear Phosphoproteins in Rice Embryos during Early Stage of Seed Germination Phosphorylation is a reversible posttranslational modification involved mainly in signal transduction and protein localization. To understand the role of nuclear phosphoproteins during rice seed germination after 0, 12, and 24 h, gel-free/label-free phosphoproteomic analysis was performed. In total, 115 nuclear phosphoproteins were identified during rice seed germination (Supplemental Table 6). After quantitative normalization of nuclear phosphoprotein abundance and one-way ANOVA, 29 nuclear phosphoproteins were identified as significantly changed in abundance during rice seed germination (Table 1, Supplemental Table 7). Based on the changes in abundance of nuclear phosphoproteins at different time points during germination, 29 nuclear phosphoproteins were divided into 5 clusters (Figure 5). Cluster 1 comprised 4 nuclear phosphoproteins that exhibited increased phosphorylation 15 ACS Paragon Plus Environment


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during germination. Cluster 2 contained 11 nuclear phosphoproteins that reached a peak phosphorylation level at 12 h of imbibition and then decreased. Clusters 3, 4, and 5 consisted of 3, 10, and 1 nuclear phosphoproteins, respectively, that had a sustained decrease in phosphorylation level. DNA and nucleotide binding proteins, zinc finger proteins, and WRC domain containing proteins, which belonged to Cluster 2, reached a peak phosphorylation level at 12 h of imbibition and then decreased (Figure 5, Table 1).

In Silico Protein-Protein Interaction Analysis of Nuclear Phosphoproteins in Rice Embryos during Early Stage of Seed Germination To determine which of the 29 nuclear phosphoproteins that significantly changed in abundance may interact during rice seed germination, time-dependent changes in the abundance of each phosphoprotein were used in conjunction with an S-system based modeling approach. After protein-protein interaction analysis, the network of these nuclear phosphoproteins was revealed (Figure 6). A total of 29 nuclear phosphoproteins were predicted to interact with other proteins. Nuclear phosphoproteins in Cluster 2 mainly induced expression of peptidase S1C_HrtA/DegP2/Q/S protein (protein No. 1) and AT-hook protein 1 (protein No. 2). Sas10/Utp3 protein (protein No. 23) was positioned in the center of network of nuclear phosphoproteins. The abundance of Sas10/Utp3 protein was suppressed by peptidase S1C HrtA/DegP2/Q/S protein and AT-hook protein 1 during rice seed germination.

RNA Expression Level of Nuclear Phosphoproteins in Rice Embryos during Early Stage of Seed Germination Among 29 nuclear phosphoproteins which exhibited significant changes in abundance during rice seed germination, 8 of them were selected for qRT-PCR to check their RNA 16 ACS Paragon Plus Environment

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expression levels in rice embryos during early stage of seed germination. These 8 nuclear phosphoproteins were both predicted to locate in nuclei using WoLF PSORT and NucPred softwares, and the ratio of protein abundance during the germination process was above or below 10 times. Eight qRT-PCR products were analyzed by agarose gel electrophoresis and the sizes of products were consistent with design (Supplemental Figure 3, numbers 2, 3, 4, 5, 6, 7, 8, and 9). Among 8 examined genes, the mRNA expression levels of 5 genes including nucleotide binding protein, WRC domain containing protein, multidomain cyclophilin type peptidyl prolyl cis-trans isomerase, GTP binding signal recognition particle SRP54 G-domain containing protein, and Sas10/Utp3 protein did not change in rice embryos during early stage of seed germination. However, the mRNA expression level of AT hook DNA binding protein was up-regulated in rice embryos at 12 h and down-regulated at 24 h of imbibition. The mRNA expression level of zinc finger CCCH type protein was down-regulated in rice embryos during early stage of seed germination. In contrast, the mRNA expression level of ribosomal RNA processing protein 7 was up-regulated in rice embryos during early stage of seed germination (Figure 7).

Analysis of the Role of Zinc Finger Proteins and Sas10/Utp3 Protein in Rice Embryos during Early Stage of Seed Germination In 29 nuclear phosphorylated proteins predicted interaction network, two zinc finger proteins which are zinc finger CCCH type and zinc finger BED type located on the upstream of network to interact with other proteins (Figure 6). In Arabidopsis, some zinc finger proteins were confirmed that they involved in seed germination through the interaction between zinc finger proteins and phytohormones such as GA and ABA 40,41. To investigate the role of phosphorylation in rice embryos during early stage of seed germination, the effect of phosphatase inhibitor on seed germination was examined. 17 ACS Paragon Plus Environment


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Furthermore, the mRNA expression levels of two zinc finger proteins were analyzed in rice embryos during early stage of seed germination with phytohormones and phosphatase-inhibitor treatments. Rice seeds were treated with phosphatase inhibitor, GAs, and ABA for 24, 48, and 72 h of imbibition. The phenotypes of seed germination were similar between phosphatase inhibitor and ABA treatments (Figure 8A). The germination rate of seeds treated with phosphatase inhibitor was significantly slow down (Figure 8A). The water uptake of seeds treated with phosphatase inhibitor or ABA was significantly lower than that of control (Figure 8B). The mRNA expression level of zinc finger CCCH type protein was down-regulated in rice embryos during early stage of seed germination with phosphatase-inhibitor treatment (Figure 9A). In contrast, the mRNA expression level of zinc finger BED type protein was up-regulated in rice embryos during early stage of seed germination with phosphatase inhibitor and ABA treatments (Figure 9B). Additionally, the mRNA expression of Sas10/Utp3 protein was analyzed (Figure 10). It was no change in rice embryos during early stage of seed germination with GAs and ABA treatments. However, it was up-regulated with phosphatase inhibitor in rice embryos during early stage of seed germination (Figure 10).

DISCUSSION Seed germination, which usually starts with the uptake of water by dry seed and terminates with elongation of radicle 2,3. Rice seeds were germinated on moist mud bed in agriculture and wet filter paper in experiment 7,27. In this study, some hypoxic inducible proteins were detected by proteomic analysis for total proteins (Supplemental Table 2). In both germination conditions, some part of seeds but not the whole seeds were submerged in water. It indicated that rice can germinate in low oxygen condition 42. Howell et al. 15,16 18 ACS Paragon Plus Environment

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characterized changes in water content and metabolic activity in embryo during rice seed germination under aerobic and anaerobic conditions, and furthermore Narsai et al. 43 defined 10 metabolites and 1136 transcripts as aerobic responders, and 13 metabolites and 730 transcripts as anaerobic responders in rice embryos and young seedlings. In order to assess whether seeds germinate on wet filter paper are under anaerobic condition, the total proteins identified in this nuclear phosphoproteomics were compared with transcriptomic data in research by Narsai et al. 43. In 349 total proteins, there are 8 and 4 genes which encoding proteins were identified in anaerobic and aerobic responders respectively (Supplemental Table 8). It might indicate that there is hypoxic stress in rice embryos during early stage of seed germination to some extent. However, it might because at the early stage of germination, anaerobic respiration is the major source of ATPs 44,45. Seed germination is a well-regulated process including a series of signal transduction and gene expression steps 46. The first 24 h of rice seed imbibition is usually regarded as the most important stage because germination-related metabolic reactions are reactivated during this period 3. Transcriptomic analysis of germinating rice seeds indicated that the onset of mRNA biosynthesis starts at an early stage of germination 4. To better understand the phosphorylation of nuclear proteins in rice embryos at this stage, the phosphorylation status of nuclear proteins was analyzed. In this study, 22 transcription factors were identified as nuclear phosphoproteins expressed in rice embryos after 24 h of imbibition (Supplemental Table 5). Phosphoproteomics of total protein indicated that a number of transcription factors change phosphorylation status in rice embryos during the early stage of seed germination 17. However, most of the transcription factors identified as phosphoproteins in nucleus (supplemental table 6) in this study didn’t exist in previous study 17. It indicated that subcellular proteomic analysis of the nucleus during rice seed germination is useful. Of the transcription factors, two AT-hook DNA binding proteins, which were AT-hook 19 ACS Paragon Plus Environment


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DNA binding protein 1 (Os08t0512400-02) and AT-hook DNA binding conserved site domain containing protein (Os04t0501600-01), were identified in the nucleus and demonstrated significantly increased phosphorylation levels after 12 h of imbibition, which then dramatically decreased (Table 1, Figure 5). AT-hook DNA binding proteins are AT-rich DNA binding proteins belonging to the high mobility group non-histone chromosomal protein HMG-I(Y) family 47. In plants, an AT-hook motif was found to bind to special regions of chromosomal DNA called nuclear matrix attachment regions 48. In Arabidopsis, an AT-hook protein called AGF1 responds to the GA-controlled negative feedback of AtGA3ox1, encoding GA3-oxidase, at the posttranscriptional or posttranslational level 49. In this study, two AT-hook DNA binding proteins had phosphorylation sites (Supplemental Table 4) and the abundance of these proteins were increased after 12 h of imbibition and then dramatically decreased. These results suggest that they might be involved in the homeostasis of GAs through changing protein phosphorylation status during rice seed germination. Based on analysis of in silico protein-protein interactions of 29 nuclear phosphoproteins, Sas10/Utp3 was interacted with many proteins and it might be positioned in the center of the network of nuclear phosphoproteins. Furthermore, its abundance was suppressed by peptidase S1C HrtA/DegP2/Q/S (Os06t0234100) protein and AT-hook protein 1 during germination (Figure 6). Peptidase S1C HrtA/DegP2/Q/S, which contains a recognizable PDZ domain, functions in mRNA export from the nucleus and protein import into the nucleus 50,51. In Arabidopsis, rice, and poplar, the HrtA/DegP2 family contains 16, 15, and 17 homolog proteins, respectively 52-54. However, only one DegP protein (Deg9, At5g40200) has been identified as a nuclear protein experimentally and by a software algorithm in Arabidopsis 55-57, and it has two orthologs (Os02t0742500 and Os06t0234100) in rice 51. It has been hypothesized that this protein is involved in ribosomal DNA 20 ACS Paragon Plus Environment

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transcription and ribosomal RNA processing/modification 50. In this study, peptidase S1C HrtA/DegP2/Q/S protein demonstrated an increased phosphorylation level during germination, which indicates that DegP protein in the nucleus of rice embryos might be involved in seed germination. Sas10/Utp3 protein contains a Sas10/Utp3 domain and is a component of the U3 ribonucleoprotein complex. Furthermore, it is a regulator of chromatin silencing in yeast 58 and is involved in 18S ribosomal RNA biogenesis 59. CANu1 protein, which contains a Sas10/Utp3 domain, was observed to be localized in nuclei in humans 60. This is consistent with our results using two types of software that predicted localization of Sas10/Utp3 protein in nuclei in rice embryos during germination (Table 1). However, there are not many studies of Sas10/Utp3 protein in plants. This study identified Sas10/Utp3 protein as positioned in the center of the phosphorylation network in nuclei of rice embryos (Figure 5), and the mRNA expression level of Sas10/Utp3 didn’t change in embryos during early stage of seed germination. Furthermore, the mRNA expression level of Sas10/Utp3 did not change with phytohormones, but it was up-regulated with phosphatase inhibitor in rice embryos during early stage of seed germination (Figure 10). It is suggested that Sas10/Utp3 protein might play through its phosphorylation and dephosphorylation switching during the early stage of seed germination without the relation to phytohormone. In this study, two zinc finger proteins exhibited high phosphorylation levels after 12 h of imbibition and then decreased (Table 1, Figure 5). Zinc finger proteins containing conserved cysteine and histidine ligands were first recognized in Xenopus transcription factor IIIA 61. These proteins have various functions ranging from DNA binding and RNA binding to protein-protein interactions and membrane association 62. OsLOL1, a C2C2 type zinc finger protein, promotes seed germination through increasing the GA level in rice. OsKO2, an important biosynthetic enzyme in GA biosynthesis, is upregulated through 21 ACS Paragon Plus Environment


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OsLOL1, which interacts with OsbZIP58 during germination 41. AtTZF4/5/6, CCCH zinc finger proteins, are reportedly involved in seed germination in Arabidopsis. AtTZF4/5/6 are up-regulated by ABA and downregulated by GA, and their proteins also act as a positive regulator for ABA- and negative regulator for GA-mediated seed germination responses 40. In this study, the mRNA expression level of zinc finger CCCH type protein (Os02t0161200-01) did not change in rice embryos during early stage of seed germination with phosphatase inhibitor and ABA treatments (Figure 9A). Zinc finger BED domain containing protein were thought to function as either transcription activators or repressors by modifying local chromatin structure on binding to GC-rich sequences 63. It also was reported that zinc BED type proteins involve in potato and barley disease resistance 64,65. However, there is no report about zinc finger BED type protein in seed during germination. In this study, the mRNA expression level of zinc finger BED type protein (Os03t0733400-01) was up-regulated in rice embryos during early stage of seed germination with phosphatase inhibitor and ABA treatments (Figure 9B). It might be because seed needs more dephosphorylating status of zinc finger BED type proteins to promote germination. These results indicate that zinc finger BED type and CCCH type proteins might mediate rice seed germination through phosphorylation and posttranslational modifications.

CONCLUSIONS The early stage of seed germination is the first step in rice life cycle without visible morphological change 3. To investigate the mechanism controlling the physiological status shifting in the early stage of rice seed germination, nuclear phosphoproteomics was performed. The main findings of this nuclear phosphoproteomics are as follows: (i) Out of 115 identified nuclear phosphoproteins, 29 nuclear phosphoproteins significantly changed 22 ACS Paragon Plus Environment

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the phosphorylation level during early stage of seed germination; (ii) Study of in silico protein-protein interactions for 29 nuclear phosphoproteins indicated that Sas10/Utp3 protein was interacted with most phosphoproteins in nuclei, and its mRNA expression level didn’t change in rice embryos during early stage of seed germination; (iii) Zinc finger proteins which are zinc finger CCCH type protein and zinc finger BED type protein play important roles through their phosphorylation/dephosphorylation during early stage of seed germination. To investigate the role of phosphorylated status of zinc finger proteins and Sas10/Utp3 proteins in rice embryos during early stage of seed germination, the effect of phosphatase inhibitor on seed germination was examined. Physiological experiments indicated that protein dephosphorylation might promote rice seed germination. Taken together, it is suggested that phosphorylation and dephosphorylation of nuclear proteins involve in rice seed germination, and transcription factors such as zinc finger CCCH type protein and zinc finger BED type protein might have key role through phosphorylation/dephosphorylation. Furthermore, Sas10/Utp3 protein might be interacted with most nuclear phosphoproteins in rice embryos to mediate the early stage of seed germination.

ASSOCIATED CONTENT Supporting Information Supplemental Table 1. Primer sequences of genes selected for qRT-PCR. Supplemental Table 2. Total proteins identified in rice embryos after 24 h of imbibition. Supplemental Table 3. Nuclear proteins identified in rice embryos after 24 h of imbibition. Supplemental Table 4. The probabilities of phosphorylation site assignment for nuclear phosphopeptides in rice embryos after 24 h of imbibition. Supplemental Table 5. Nuclear phosphoproteins identified in rice embryos after 24 h of 23 ACS Paragon Plus Environment


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imbibition. Supplemental Table 6. Nuclear phosphoproteins identified in rice embryos during early stage of germination. Supplemental Table 7. The probabilities of phosphorylation site assignment for nuclear phosphopeptides in rice embryos during early stage of germination. Supplemental Table 8. Comparison of the proteome data in this study with transcriptome data in Narsai et al. 43 in rice embryo. Supplemental Figure 1. Experimental design of nuclear phosphoproteomic study. Supplemental Figure 2. Procedure for isolation of nuclei. Supplemental Figure 3. Agarose gel electrophoresis of qRT-PCR products. This material is available free of charge via the Internet at http://pubs.acs.org.

Accession Codes The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository 66

with the dataset identifier PXD001774.

AUTHOR INFORMATION Corresponding Author * (S.K.) Tel: +81-29-838-8693. Fax: +81-29-838-8694. E-mail: [email protected]. Author Contribution # M.L. and X.Y. contributed equally to this work.

Notes The authors declare no competing financial interest. 24 ACS Paragon Plus Environment

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ACKNOWLEDGMENTS The authors thank Dr. K. Sugimoto at the National Institute of Agrobiological Sciences for providing rice seeds. The authors thank Dr. Y. Nanjo and Dr. K. Nishizawa at the National Institute of Crop Science for useful suggestion and comments. The authors also thank Ms. C. Kawashima and Ms. A. Sato at the National Institute of Crop Science for experimental support during this research. This work was supported by the National Natural Science Foundation of China (31271805) and by the National Agriculture and Food Research Organization.

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Figure legends Figure 1. Western blot analysis of nuclear proteins purified from rice embryos. Total (TP) and nuclear (N) proteins were purified from rice embryos after 24 h of imbibition. Proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with anti-histone or ascorbate peroxidase antibodies. Anti-rabbit IgG conjugated with horseradish peroxidase was used as the secondary antibody. Signals were detected using an ECL system. “M” denotes the marker lane. Coomassie brilliant blue (CBB) staining of the gel was used as a loading control and the relative intensities of bands were calculated. Data are means ± SD from 3 independent biological replicates (R1, R2, and R3). Asterisks indicate significant changes according to Student’s t-test (*P

Proteomic Analysis of Phosphoproteins in the Rice Nucleus During the

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