Article pubs.acs.org/jpr
Quantitative Proteomics Reveals the Role of Protein Phosphorylation in Rice Embryos during Early Stages of Germination Chao Han,†,‡,§ Pingfang Yang,*,† Katsumi Sakata,∥ and Setsuko Komatsu*,‡ †
Key Laboratory of Plant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Moshan, Wuchang, Wuhan 430074, China ‡ National Institute of Crop Science, National Agriculture and Food Research Organization, 2-1-18 Kannondai, Tsukuba 305-8518, Japan § University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China ∥ Maebashi Institute of Technology, 460-1 Kamisadorimachi, Maebashi 371-0816, Japan S Supporting Information *
ABSTRACT: Seed germination begins with water uptake and ends with radicle emergence. A gel-free phosphoproteomic technique was used to investigate the role of protein phosphorylation events in the early stages of rice seed germination. Both seed weight and ATP content increased gradually during the first 24 h following imbibition. Proteomic analysis indicated that carbohydrate metabolism- and protein synthesis/degradationrelated proteins were predominantly increased and displayed temporal patterns of expression. Analyses of cluster and protein− protein interactions indicated that the regulation of sucrose synthases and alpha-amylases was the central event controlling germination. Phosphoproteomic analysis identified several proteins involved in protein modification and transcriptional regulation that exhibited significantly temporal changes in phosphorylation levels during germination. Cluster analysis indicated that 12 protein modification-related proteins had a peak abundance of phosphoproteins at 12 h after imbibition. These results suggest that the first 12 h following imbibition is a potentially important signal transduction phase for the initiation of rice seed germination. Three core components involved in brassinosteroid signal transduction displayed significant increases in phosphoprotein abundance during the early stages of germination. Brassinolide treatment increased the rice seed germination rate but not the rate of embryonic axis elongation. These findings suggest that brassinosteroid signal transduction likely triggers seed germination. KEYWORDS: phosphorylation, proteomics, rice, seed germination
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INTRODUCTION Seed germination, which begins with water imbibition and ends with radicle extrusion,1 is an essential step in the initiation of a new life cycle in plants. Completion of this step requires the coordination of several signal transduction and biochemical processes, including those involved in responding to environmental factors and mobilizing seed reserves, finally leading to elongation of the embryonic axis.2 The seed germination process is predominantly controlled by phytohormones, including gibberellins (GAs) and abscisic acid (ABA), which play antagonistic roles as positive and negative regulators, respectively, during germination.3,4 The seed germination process is largely programmed during seed maturation.2 In mature Arabidopsis seeds, numerous stored mRNAs are specifically involved in initiating germination.5 The translation of stored mRNA during maize germination is mediated by the initiation factor eIFiso4E.6 The importance of stored mRNA for the germination process was demonstrated in Arabidopsis, as the double mutant i4gl/i4g2 exhibited a low germination rate and poor seed viability.7 In addition, it was © 2014 American Chemical Society
demonstrated that treatment with the translation inhibitor cycloheximide completely blocked seed germination of both Arabidopsis and rice and that treatment with the transcription inhibitor α-amanitin strongly affected the germination rate but did not prevent radicle protrusion.8,9 These findings indicate that stored transcripts are more vital for germination than de novo synthesized transcripts. Supporting this conclusion, newly synthesized proteins radiolabeled with 35S-methionine strongly increased after 8−24 h in Arabidopsis compared with those synthesized from 0 to 8 h during germination, indicating that the translation of stored mRNA occurred during the early stages of germination and was followed later by protein de novo synthesis.10 Reversible modification of proteins by phosphorylation is critical for the regulation of signal transduction in plants and other organisms. Many protein kinases and phosphatases participate in ABA signaling to regulate seed germination.11 For Received: December 27, 2013 Published: January 24, 2014 1766
dx.doi.org/10.1021/pr401295c | J. Proteome Res. 2014, 13, 1766−1782
Journal of Proteome Research
Article
collected for measurement of the ATP content. After the addition of 100 μL of the supernatant sample into 1 mL reaction solution (280 mM HEPES-NaOH, 4 mM MgCl2, 2 mM glucose, 2 mM NAD, and 4 U/mL glucose-6-phosphate dehydrogenase), the absorbance at 340 nm was measured using a DU730 spectrophotometer (Beckman, Indianapolis, IN). Hexokinase (4 U/mL) was added to the reaction solution, which was then incubated at 25 °C for 30 min, and the absorbance at 340 nm was again measured. ATP content was determined by measuring the increase in absorbance at 340 nm before and after the addition of hexokinase.
example, type 2C protein phosphatases and SNF1-related protein kinase 2 are core members of the four-component signaling system of the ABA signaling pathway.12,13 In addition, mitogen-activated protein kinase (MAPK) was reported to arrest Arabidopsis seed germination through phosphorylation of ABI5, resulting in an increase in the ABA signal strength.14 Using a phosphoproteomics strategy, ABA-induced activation of the Arabidopsis MAPKs AtMPK1 and AtMPK2 was shown to be promoted by SnRK2.15 Ca2+-dependent protein kinase (CDPK) is another type of kinase involved in ABA signaling and regulation of seed germination. AtCPK12-RNAi lines of Arabidopsis display ABA hypersensitivity during seed germination.16 Biochemical assays revealed that AtCPK12 interacts and stimulates the activity of phosphorylated ABI2 and also phosphorylates two ABA-responsive transcription factors, ABF1 and ABF4, in vitro.16 Rice seeds have a tiny embryo and relatively large endosperm used for nutrient storage. Rice germination can be divided into three phases based on water uptake: rapid water uptake (phase I), followed by a plateau phase of water uptake (phase II), and initiation of the postgerminative growth phase (phase III).1,17 A well-studied process in rice germination is the mobilization of starch in endosperm through GA-induced alpha-amylase expression. It has been shown that the ABA-induced protein kinase PKABA1 suppresses alpha-amylase expression through repression of the GA-mediated induction of MybGA, which regulates alpha-amylase expression. PKABA1 acts upstream of the formation of functional MybGA but downstream of DELLA proteins, which function as negative regulators of GA signaling.18 Kashem et al.19 showed that Ca2+ participates in the regulation of alpha-amylase expression in rice. Specifically, in the absence of exogenous Ca2+, neomycin, which is a potent inhibitor of inositol phospholipid-specific phospholipase C, markedly reduces the germination speed and seedling growth of rice seeds and inhibits the GA-induced expression of alpha-amylase.19 Phosphorylation is an important part of the ABA signal transduction pathway.11 In addition, many signal transduction events involve protein phosphorylation modification, including activation of alpha-amylase expression during rice seed germination. Thus, protein phosphorylation appears to be intimately involved in regulating the germinating process. To date, however, no systemic studies have examined the phosphoproteomics of rice seed germination. Here a gel-free phosphoproteomics approach was used to investigate the role of protein phosphorylation in rice seed germination.
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Protein Extraction and Digestion for Proteomic Analysis
A portion (50 mg) of rice embryo tissues was ground in liquid nitrogen with a mortar and pestle and mixed with an acetone solution containing 10% trichloroacetic acid and 0.07% 2-mercaptoethanol. After 1 h of incubation at −20 °C, the suspension was centrifuged and the obtained pellet was washed twice with cold acetone containing 0.07% 2-mercaptoethanol and then evaporated in a Speed-Vac (Savant Instruments, Hicksville, NY) for 10 min. The pellet was suspended in 0.5 mL of lysis buffer (7 M urea, 2 M thiourea, 5% CHAPS, and 2 mM tributylphosphine), and the protein concentration was then measured by the Bradford method20 using Protein Assay reagent (Bio-Rad, Hercules, CA). The extracted proteins (150 μg) from the rice embryo tissue were purified as follows. After the previous samples were adjusted to a volume of 150 μL, 600 μL of methanol was added and the resulting solution was 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 000g for 5 min. The upper aqueous phase was discarded, and 450 μL of methanol was added to the organic phase. The samples were centrifuged at 20 000g for 5 min, and the resulting supernatants were discarded, and the pellets were dried. The dried pellets were resuspended in 50 mM NH4HCO3, and the proteins 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 resulting peptides were desalted using a MonoSpin C18 column (GL Science, Tokyo, Japan). Protein Extraction and Digestion for Phosphoproteomic Analysis
A portion (50 mg) of rice embryo was ground in liquid nitrogen with a mortar and pestle and 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 (Sigma, St. Louis, M), and protease inhibitor mixture (Roche, Werk Penzberg, Germany). After centrifugation at 12 000g for 20 min, the supernatant was diluted with 3 volumes cold acetone containing 0.07% 2-mercaptoethanol and incubated at −20 °C for 2 h. The 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 performing purification by the same method used for the proteomic analysis, 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.
EXPERIMENTAL PROCEDURES
Plant Material and Seed Germination
Seeds of rice (Oryza sativa cultivar Nipponbare) were collected and placed in 10 cm × 10 cm culture plates for germination. Seeds were imbibed in distilled water in dark growth chamber at 25 °C and 70% relative humidity. Embryos were separated from seeds using operating knife blade and put into liquid nitrogen, then stored in −80 °C. In total, 60 and 80 seeds were prepared for ATP measurements and proteomic analysis, respectively. All experiments were repeated three times. Measurement of ATP Content
A portion (0.1 g) of rice embryo was ground in liquid nitrogen with a mortar and pestle and was then mixed with 1 mL of ATP extraction buffer (0.51 M trichloroacetic acid and 17 mM EDTA). After sonication for 10 min, the sample was centrifuged at 20 000g for 5 min, and the resulting supernatant was 1767
dx.doi.org/10.1021/pr401295c | J. Proteome Res. 2014, 13, 1766−1782
Journal of Proteome Research
Article
performed as part of the search. Mascot results were filtered with Mascot Percolator to improve the accuracy and sensitivity in the peptide identification. False discovery rates for peptide identification of all searches were 0.9 were considered to be candidate interactions and were further evaluated as model interactions.24 Interactions between significant proteins were identified based on the ‘sum of covariances’ (SUMCOV) method,25 as we selected proteins with a large value of SUMCOV, suggesting a number of interactions from the protein.
detected, although enlargement of the embryo was observed (Figure 1A). Seed weight increased rapidly during the first 6 h of imbibition, but the rate of increase slowed gradually over time (Figure 1B). In contrast, the ATP content increased slowly during the first 6 h of imbibition but then increased rapidly and reached the maximum concentration at 24 h. However, from 24 to 48 h, which was the plateau phase of rice seed germination, the ATP content decreased continuously (Figure 1C). Changes of Protein Abundance of Rice Embryos during Early Stage of Seed Germination
To explore changes in the proteome during germination, we performed gel-free proteomic analysis on rice embryos after 0, 6, 12, 24, and 48 h of imbibition. After protein identification by MS/MS, 852 proteins were significantly changed during germination based on one-way ANOVA analysis (Supplemental Table 2 in the Supporting Information). On the basis of MapMan ontology, the 852 proteins could be separated into 13 functional categories: metabolism, hormone, transport, redox, signaling, RNA, DNA, protein, cell, development, stress, miscellaneous enzyme family, and unassigned proteins. Among these categories, metabolism and protein contained the largest amount of proteins, comprising 29 and 26%, respectively, of all significantly changed proteins (Figure 2A). The metabolism functional category contained 243 proteins, among which three clusters (Clusters I, II, and III) showed marked changes in protein abundance during germination. Cluster I, which included sucrose synthase, α-amylase, and S-adenosylmethionine synthetase, displayed extremely high protein abundance from 24 to 48 h after imbibition, which was the plateau phase of rice seed germination. Sucrose synthase and α-amylase participate in starch degradation, whereas
Statistical Analysis
The statistical significance of the results was evaluated with the Student’s t test when only two groups were compared by oneway ANOVA when multiple groups were compared. All calculations were performed using Graphpad software (version 5.0). A p value of 0.9 were considered as candidate interactions. (A) Diagrammatic representation of the interactions between 848 out of the 852 significantly changed proteins. The four proteins not shown were isolated but were not found to form significant interactions. Red lines indicate inductive interactions and correspond to gij >0 in the S-system differential equation, and blue lines show suppressive interactions and correspond to gij < 0. B) Interactions between the 19 significant proteins selected based on SUMCOV statistical criterion. The 19 proteins are colored yellow in the diagram shown in panel A. MPHO17, NADH-dependent hydroxypyruvate reductase; MMAC6, sucrose synthase 3; MMAC8, sucrose synthase 2; MMAC11, sucrose synthase 2; MMAC15, alpha amylase; MMAC18, alpha amylase; MAA41, S-adenosylmethionine synthetase1; MAA42, S-adenosylmethionine synthetase1; RED16, L-ascorbate peroxidase; MISC28, Haem peroxidase; SIG29, YT521-B-like protein family protein; SIG30, YT521-B-like protein family protein; SIG31, YT521-Blike protein family protein; SIG32, YT521-B-like protein family protein; RNA38, ribonuclease T2 family protein; RNA39, RNase S-like protein; RNA 40, S-like ribonuclease; NA68, lipoxygenase-LH2 domain containing protein; and NA93, conserved hypothetical protein.
Figure 4. Phosphoproteomic analysis of rice embryos during germination. (A) Number of phosphorylation sites in phosphopeptides identified in germinating embryos. (B) Different phosphorylation sites in single-phosphorylated peptides identified in germinating embryos. (C) Functional categories of significantly changed phosphoproteins in rice embryos during germination based on MapMan ontology. (D). Subgroup distribution of protein destination-related proteins based on MapMan ontology.
serine and threonine phosphorylated peptides accounting for 74 and 21%, respectively, of these peptides. Only 20 tyrosinephosphorylated peptides were identified in the MS analysis (Figure 4B). After quantitative normalization of phosphoprotein abundance and one-way ANOVA testing, 149 phosphorylated proteins were found to have significantly changed in abundance during germination (Table 1, Supplemental Table 3 in the Supporting Information). The differentially expressed phosphorylated proteins could be divided into 12 functional categories based on MapMan ontology. Protein destination-related proteins comprised the largest functional category, and almost half of these proteins were involved in posttranslational modification and displayed kinase or phosphatase activity (Figure 4C,D). In contrast with the proteomic analysis results, metabolism-related proteins were not the most abundant phosphorylated proteins and were detected in equal number to protein members of the transport and RNA functional categories (Figure 4C). In addition, 47
Changes of Phosphoprotein Abundance in Rice Embryos during Early Stages of Seed Germination
Phosphorylation is a common, reversible, post-translational modification that is mainly involved in signal transduction and protein localization in plants and other organisms. To further explore the mechanisms that trigger seed germination, we 1771
dx.doi.org/10.1021/pr401295c | J. Proteome Res. 2014, 13, 1766−1782
similar to enoyl-ACP reductase (fragment) orthophosphate dikinase precursor (EC 2.7.9.1) similar to isoform 2 of pyruvate-phosphate dikinase 1-chloroplastic similar to enoyl-[acyl-carrier-protein] reductase [NADH]-chloroplast precursor (EC 1.3.1.9) (NADH-dependent enoyl-ACP reductase) similar to pyruvate dehydrogenase E1 component alpha subunit-mitochondrial precursor (EC 1.2.4.1) (PDHE1-A) similar to pyruvate dehydrogenase E1 alpha subunit (EC 1.2.4.1) similar to N6-adenosine-methyltransferase 70 kDa subunit (EC 2.1.1.62) (MT-A70) (methyltransferase-like protein 3) . splice isoform 2 similar to porphobilinogen deaminase (fragment) similar to porphobilinogen deaminase-chloroplastic similar to 3-(2-)-5-bisphosphate nucleotidase similar to possible apospory-associated like protein similar to phosphoribosylformylglycinamidine synthase similar to cellulose synthase-4
name
Os02g0168800-01 Os02g0168800-02 Os12g0183300-01 Os04g0658000-02 Os01g0888500-01 Os07g0208500-01 Hormone Os03g0744600-01 similar to ripening-associated protein (fragment) Miscellaneous Enzyme Family Os06g0247800-01 similar to dynamin-like protein (fragment) Os02g0738900-01 dynamin-related protein-secondary cell wall cellulose biosynthesis Os08g0359200-02 similar to lipid phosphate phosphatase 2 (EC 3.1.3.-) (AtLPP2) (phosphatidic acid phosphatase 2) (AtPAP2) (prenyl diphosphate phosphatase) Os08g0359200-03 similar to lipid phosphate phosphatase 3 Signaling Os05g0465100-01 RabGAP/TBC domain containing protein Os01g0135700-01 EF-HAND 2 domain containing protein Os03g0263900-01 EF-HAND 2 domain containing protein Os07g0145400-01 protein kinase- core domain containing protein Transport Os09g0346400-01 conserved hypothetical protein Os09g0346400-02 hypothetical conserved gene Os05g0345100-00 similar to patellin-5 Os11g0648000-01 similar to Na-/H- antiporter Os11g0648000-02 similar to sodium/hydrogen exchanger Os09g0346400-01 similar to hexose transporter Os09g0346400-02 ABC transporter-transmembrane domain containing protein Os05g0345100-00 similar to conserved NnrU/NnuR ortholog membrane enzyme Os11g0648000-01 similar to pleiotropic drug resistance protein 3 Os11g0648000-02 similar to PDR-like ABC transporter Os10g0539900-04 similar to PDR-type ABC transporter 2 (fragment) Os06g0561800-00 similar to PDR-like ABC transporter (PDR4 ABC transporter) Os12g0405200-01 similar to PDR20
Os02g0739600-01 Os06g0246500-01 Os02g0672600-01
Metabolism Os08g0327400-01 Os05g0405000-01 Os05g0405000-02 Os09g0277800-01
gene ID
Table 1. Changes in Phosphoprotein Abundance during Rice Seed Germination
1 1 1 1 3 3 2 2 7 7 5 2 2 2 1 1 1 1 1 1 1
1 3 3 2 2 7 7 5 2 2 2 1 1 1 1 1 1 1
3
6 2 2 1
2 2 1 1 1 1
3 3 3
2 1 1 2
PPb
2 2 2 2 1 1
4 4 3
7 6 6 4
MPa
1772
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
1.00 1.00 1.00 1.00
1.00
1.00 1.00 1.00
1.00
1.00 1.00 1.00 1.00 1.00 1.00
1.00 1.00 1.00
1.00 1.00 1.00 1.00
0h
6.40 6.40 1.98 10.11 10.11 112.40 121.98 3.11 1.39 1.39 1.39 1.39 1.39
315.04 18.14 2.93 1.46
10.25
3.59 0.39 10.25
85.72
2.04 2.04 21.23 1.55 8.51 0.02
0.41 0.41 3.73
0.87 0.20 0.20 1.44
42.94 42.94 29.83 15.84 15.84 168.81 255.77 10.73 1.17 1.17 1.17 1.17 1.17
437.79 56.35 5.34 51.30
26.50
13.31 0.23 26.50
59.46
3.38 3.38 110.28 20.79 17.07 0.10
0.04 0.04 23.03
1.05 0.63 0.63 1.29
12 h
49.92 49.92 7.92 8.57 8.57 7.51 394.05 9.97 10.50 10.50 10.50 10.50 10.50
346.95 8.08 4.14 0.00
16.81
4.22 0.66 16.81
63.03
2.17 2.17 59.99 0.61 9.18 1.43
1.93 1.93 11.32
1.15 0.78 0.78 2.32
24 h
phosphoprotein abundance 6h
96.95 96.95 59.15 9.46 9.46 60.40 313.26 65.02 36.36 36.36 36.36 36.36 36.36
168.07 1.26 3.40 8.72
3.74
476.48 170.03 3.74
118.94
0.93 0.93 861.54 27.79 2.98 37.66
77.56 77.56 11.83
31.37 136.44 136.44 23.65
48 h
