Large Scale Identification and Quantitative Profiling of Phosphoproteins Expressed during Seed Filling in Oilseed Rape

Seed filling is a dynamic, temporally regulated phase of seed development that determines the composition of storage reserves in mature seeds. Although the metabolic pathways responsible for storage reserve synthesis such as carbohydrates, oils, and proteins are known, little is known about their regulation. Protein phosphorylation is a ubiquitous form of regulation that influences many aspects of dynamic cellular behavior in plant biology. Here a systematic study has been conducted on five sequential stages (2, 3, 4, 5, and 6 weeks after flowering) of seed development in oilseed rape (Brassica napus L. Reston) to survey the presence and dynamics of phosphoproteins. High resolution two-dimensional gel electrophoresis in combination with a phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescence stain revealed ∼300 phosphoprotein spots. Of these, quantitative expression profiles for 234 high quality spots were established, and hierarchical cluster analyses revealed the occurrence of six principal expression trends during seed filling. The identity of 103 spots was determined using LC-MS/MS. The identified spots represented 70 non-redundant phosphoproteins belonging to 10 major functional categories including energy, metabolism, protein destination, and signal transduction. Furthermore phosphorylation within 16 non-redundant phosphoproteins was verified by mapping the phosphorylation sites by LC-MS/MS. Although one of these sites was postulated previously, the remaining sites have not yet been reported in plants. Phosphoprotein data were assembled into a web database. Together this study provides evidence for the presence of a large number of functionally diverse phosphoproteins, including global regulatory factors like 14-3-3 proteins, within developing B. napus seed. Seed filling is a dynamic, temporally regulated phase of seed development that determines the composition of storage reserves in mature seeds. Although the metabolic pathways responsible for storage reserve synthesis such as carbohydrates, oils, and proteins are known, little is known about their regulation. Protein phosphorylation is a ubiquitous form of regulation that influences many aspects of dynamic cellular behavior in plant biology. Here a systematic study has been conducted on five sequential stages (2, 3, 4, 5, and 6 weeks after flowering) of seed development in oilseed rape (Brassica napus L. Reston) to survey the presence and dynamics of phosphoproteins. High resolution two-dimensional gel electrophoresis in combination with a phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescence stain revealed ∼300 phosphoprotein spots. Of these, quantitative expression profiles for 234 high quality spots were established, and hierarchical cluster analyses revealed the occurrence of six principal expression trends during seed filling. The identity of 103 spots was determined using LC-MS/MS. The identified spots represented 70 non-redundant phosphoproteins belonging to 10 major functional categories including energy, metabolism, protein destination, and signal transduction. Furthermore phosphorylation within 16 non-redundant phosphoproteins was verified by mapping the phosphorylation sites by LC-MS/MS. Although one of these sites was postulated previously, the remaining sites have not yet been reported in plants. Phosphoprotein data were assembled into a web database. Together this study provides evidence for the presence of a large number of functionally diverse phosphoproteins, including global regulatory factors like 14-3-3 proteins, within developing B. napus seed. The storage reserves found in most plant seeds consist of carbohydrates, oils, and proteins (1Norton G. Harris J.F. Compositional changes in developing rape seed (Brassica napus L.).Planta. 1975; 123: 163-174Crossref PubMed Scopus (121) Google Scholar, 2Mienke D.W. Chen J. Beachy R.N. Expression of storage-protein genes during soybean seed development.Planta. 1981; 153: 130-139Crossref PubMed Scopus (150) Google Scholar, 3Murphy D.J. Cummins I. Kang A.S. Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation.Biochem. J. 1989; 258: 285-293Crossref PubMed Scopus (89) Google Scholar, 4Baud S. Boutin J. Miquel M. Lepiniec L. Rochat C. An integrated overview of seed development in Arabidopsis thaliana ecotype WS.Plant Physiol. Biochem. 2002; 40: 151-160Crossref Scopus (363) Google Scholar, 5Hills M.J. 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Beachy R.N. Expression of storage-protein genes during soybean seed development.Planta. 1981; 153: 130-139Crossref PubMed Scopus (150) Google Scholar). Numerous studies including two global approaches, transcriptomics and proteomics, conducted to date on seed filling, in particular oilseed plants such as oilseed rape (Brassica napus L.), soybean, and Arabidopsis, continue to produce new paradigms and refine our understanding of the existing biosynthetic pathways responsible for accumulation of seed storage reserves (5Hills M.J. Control of storage-product synthesis in seeds.Curr. Opin. Plant Biol. 2004; 7: 302-308Crossref PubMed Scopus (115) Google Scholar, 9Schwender J. Ohlrogge J. Shachar-Hill Y. Understanding flux in plant metabolic networks.Curr. Opin. Plant Biol. 2004; 7: 309-317Crossref PubMed Scopus (136) Google Scholar, 10Weber H. Ljudmilla B. Wobus U. Molecular physiology of legume seed development.Annu. Rev. 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Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.Plant Physiol. 2005; 137: 1397-1419Crossref PubMed Scopus (311) Google Scholar). A microarray of developing Arabidopsis seed provided insight into primary transcriptional networks that coordinate the metabolic responses to seed developmental programs and lead to the distribution of carbon among carbohydrate, oil, and protein reserves (12Ruuska S.A. Girke T. Benning C. Ohlrogge J.B. Contrapuntal networks of gene expression during Arabidopsis seed filling.Plant Cell. 2004; 14: 1191-1206Crossref Scopus (464) Google Scholar). Proteomics studies have also been performed in Medicago truncatula (18Gallardo K. Le Signor C. Vandekerckhove J. Thompson R.D. Burstin J. Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation.Plant Physiol. 2003; 133: 664-682Crossref PubMed Scopus (219) Google Scholar), pea (19Schiltz S. Gallardo K. Huart M. Negroni L. Sommerer N. Burstin J. Proteome reference maps of vegetative tissues in pea. An investigation of nitrogen mobilization from leaves during seed filling.Plant Physiol. 2004; 135: 2241-2260Crossref PubMed Scopus (118) Google Scholar), and soybean (20Hajduch M. Ganapathy A. Stein J.W. Thelen J.J. A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.Plant Physiol. 2005; 137: 1397-1419Crossref PubMed Scopus (311) Google Scholar) using high resolution two-dimensional gel electrophoresis (2-DGE) 1The abbreviations used are: 2-DGE, two-dimensional gel electrophoresis; CBB, Coomassie Brilliant Blue; CV, coefficient of variation; FA, formic acid; PGK, phosphoglycerate kinase; Pro-Q DPS, Pro-Q Diamond phosphoprotein stain; WAF, weeks after flowering; TEMED, N,N,N′,N′-tetramethylethylenediamine; 2-D, two-dimensional; ACP, acyl-carrier-protein. in combination with either MALDI-TOF-MS or LC-MS/MS. Of these studies, the soybean investigation was the most systematic with respect to quantitative expression profiling and protein identification. In that study, 679 and 422 protein spots were profiled and identified, respectively, representing 216 non-redundant proteins and 14 functional classes (20Hajduch M. Ganapathy A. Stein J.W. Thelen J.J. A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.Plant Physiol. 2005; 137: 1397-1419Crossref PubMed Scopus (311) Google Scholar). Despite these investigations, including the large data sets of genes and proteins, the specific underlying regulatory mechanisms that control the levels of storage reserves in the seed remain largely unknown. Understanding the underlying regulatory mechanism(s) of the networks by identifying and characterizing their regulatory components will undoubtedly broaden our knowledge on how the levels of stored components in the seed are fine tuned. Reversible phosphorylation is a major post-translational mechanism by which cells transduce cellular, developmental, and environmental signals and thereby control a myriad of biological processes in diverse organisms including plants (for reviews, see Refs. 21Huber S.C. Hardin S.C. Numerous posttranslational modifications provide opportunities for the intricate regulation of metabolic enzymes at multiple levels.Curr. Opin. Plant Biol. 2004; 7: 318-322Crossref PubMed Scopus (77) Google Scholar, 22Pawson T. Scott J.D. Protein phosphorylation in signaling—50 years and counting.Trends Biochem. Sci. 2005; 30: 286-290Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar, 23Mukherji M. Phosphoproteomics in analyzing signaling pathways.Expert Rev. Proteomics. 2005; 2: 117-128Crossref PubMed Scopus (33) Google Scholar). 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Hence understanding the dynamics of this post-translation modification in response to cellular as well as environmental cues can lead to the identification of candidate regulatory proteins (protein kinases) and their substrates and thereby can help in dissecting the signaling and metabolic networks. This emerging new area of systems biology is termed phosphoproteomics (34Mann M. Jensen O.N. Proteomic analysis of post-translational modifications.Nat. Biotechnol. 2003; 21: 255-261Crossref PubMed Scopus (1636) Google Scholar, 35Laugesen S. Bergoin A. Rossignol M. Deciphering the plant phosphoproteome: tools and strategies for a challenging task.Plant Physiol. Biochem. 2004; 42: 929-936Crossref PubMed Scopus (31) Google Scholar, 36Reinders J. Sickmann A. State-of-the-art in phosphoproteomics.Proteomics. 2005; 5: 4052-4061Crossref PubMed Scopus (301) Google Scholar). Many advances in phosphoproteomics technologies, including enrichment, detection, phosphorylation site mapping, and quantification of phosphoproteins, have made the large scale study of phosphoproteins a feasible task (23Mukherji M. Phosphoproteomics in analyzing signaling pathways.Expert Rev. Proteomics. 2005; 2: 117-128Crossref PubMed Scopus (33) Google Scholar, 34Mann M. Jensen O.N. Proteomic analysis of post-translational modifications.Nat. Biotechnol. 2003; 21: 255-261Crossref PubMed Scopus (1636) Google Scholar, 35Laugesen S. Bergoin A. Rossignol M. Deciphering the plant phosphoproteome: tools and strategies for a challenging task.Plant Physiol. Biochem. 2004; 42: 929-936Crossref PubMed Scopus (31) Google Scholar, 36Reinders J. Sickmann A. State-of-the-art in phosphoproteomics.Proteomics. 2005; 5: 4052-4061Crossref PubMed Scopus (301) Google Scholar, 37Salih E. Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.Mass Spectrom. Rev. 2005; 24: 828-846Crossref PubMed Scopus (107) Google Scholar, 38Kange R. Selditz U. Granberg M. Lindberg U. Ekstrand G. Ek B. Gustafsson M. Comparison of different IMAC techniques used for enrichment of phosphorylated peptides.J. Biomol. Tech. 2005; 16: 91-103PubMed Google Scholar, 39Loyet K.M. Stults J.T. Arnott D. Mass spectrometric contributions to the practice of phosphorylation site mapping through 2003.Mol. Cell. Proteomics. 2005; 4: 235-245Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). One of the major developments in this area is the detection of phosphoproteins using a unique fluorescence dye, Pro-Q Diamond phosphoprotein stain (Pro-Q DPS; Ref. 40Steinberg T.H. Agnew B.J. Gee K.R. Leung W.-Y. Goodman T. Schulenberg B. Hendrickson J. Beechem J.M. Haugland R.P. Patton W.F. Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology.Proteomics. 2003; 3: 1128-1144Crossref PubMed Scopus (336) Google Scholar). Two major advantages of this stain are: 1) it can be used for global quantitative analysis of phosphoproteins as it binds directly to the phosphate moiety of phosphoproteins with high sensitivity and linearity regardless of phosphoamino acid and 2) the stain is fully compatible with other staining methods and modern MS. Despite these recent advancements, phosphoproteins in plants have been rarely studied on a large scale basis (for a review, see Ref. 41Agrawal G.K. Yonekura M. Iwahashi Y. Iwahashi H. Rakwal R. System, trends and perspectives of proteomics in dicot plants. Part III: Unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships.J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2005; 815: 137-145Crossref PubMed Scopus (49) Google Scholar). One example of a large scale phosphoproteomics study is the identification of more than 300 phosphorylation sites from Arabidopsis plasma membrane proteins (42Nuhse T.S. Stensballe A. Jensen O.N. Peck S.C. Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database.Plant Cell. 2004; 16: 2394-2405Crossref PubMed Scopus (410) Google Scholar). To our knowledge, no large scale study of phosphoproteins has been carried out on developing plant seeds. We have embarked on large scale phosphoproteomics study during seed filling in B. napus with the following objectives: (i) to obtain quantitative expression profiles of phosphoproteins through seed development, (ii) to generate a 2-DGE phosphoprotein reference map, (iii) to determine the phosphorylation sites of phosphoproteins, and (iv) to begin building resources for dissecting biological processes that might be regulated by reversible phosphorylation, including metabolism. A high throughput 2-DGE approach in combination with Pro-Q DPS and LC-MS/MS were applied on five sequential stages 2, 3, 4, 5, and 6 weeks after flowering (WAF), covering the majority of seed filling. This study reports major achievements toward fulfilling these objectives by the establishment of a high resolution 2-DGE phosphoprotein reference map comprising 234 quantitative expression profiles, 103 identified phosphoproteins, and a map of phosphorylation site in 16 non-redundant phosphoproteins. This study also extends the oilseed proteomics web-based database with a "Brassica phosphoproteomics" resource for the plant community. Pro-Q DPS (product number P33301) and PeppermintStick phosphoprotein molecular weight standards (hereafter called PeppermintStick standards; product number P33350) were from Molecular Probes (Eugene, OR). A protein wide range SigmaMarker (hereafter called SigmaMarker; 6.5–205.0 kDa) was from Sigma (catalog number M4038). Acetic acid, acetone, acetonitrile, acrylamide, ammonium persulfate, DTT, methanol, N,N′-methylene bisacrylamide, phenol, sodium acetate, SDS, and TEMED were from Fisher Scientific. Immobiline IPG (pH 4–7 and 3–10, 24-cm) strips were obtained from GE Healthcare. All other reagents used in this study were of analytical grade. B. napus (var. Reston) seeds were grown in soil (Promix, Quakertown, PA) in a growth chamber (light/dark cycles of 16 h (23 °C)/8 h (20 °C), 48% humidity, and light intensity of 8000 lux). Plants were fertilized at 2-week intervals (all purpose fertilizer, 15:30:15, nitrogen-phosphorus-potassium). Flowers were tagged immediately after opening of buds (between 1 and 3 p.m.). Harvesting of developing seeds was also performed between 1 and 3 p.m. during the daytime at precisely 2, 3, 4, 5, and 6 WAF. At each developmental stage, the dry weight and total protein content of whole seeds were also measured. Total protein was quantified in triplicate using dye-binding protein assay kit (Bio-Rad) and chicken γ-globulin as standard. Total seed protein was prepared from each developmental stage according to a modified phenol-based procedure as described previously (20Hajduch M. Ganapathy A. Stein J.W. Thelen J.J. A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.Plant Physiol. 2005; 137: 1397-1419Crossref PubMed Scopus (311) Google Scholar). The protein pellet, obtained from the above extraction procedure, was suspended in IEF extraction buffer (8 m urea, 2 m thiourea, 2% (w/v) CHAPS, 2% (v/v) Triton X-100, 50 mm DTT) and vortexed at low speed for 30 min at room temperature followed by centrifugation at 14,000 rpm for 15 min to remove insoluble material. Supernatant was used for measuring protein concentration and 2-DGE analysis. 2-DGE was carried out using IPG strips (pH 3–10 or 4–7, 24 cm; GE Healthcare) as described previously (20Hajduch M. Ganapathy A. Stein J.W. Thelen J.J. A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.Plant Physiol. 2005; 137: 1397-1419Crossref PubMed Scopus (311) Google Scholar). One milligram of total protein was mixed with 2.25 μl of IPG buffer (pH 3–10 or 4–7; GE Healthcare) and IEF extraction buffer to bring up to 450 μl, vortexed for 30 s, and subjected to IEF followed by SDS-PAGE. A total of six to eight high resolution 2-D gels were run for each developmental stage using protein isolated from four independent biological samples to finally select four gels for further analysis. Additionally "reference gels" were also run for the purpose of spot matching and their downstream analysis (20Hajduch M. Ganapathy A. Stein J.W. Thelen J.J. A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.Plant Physiol. 2005; 137: 1397-1419Crossref PubMed Scopus (311) Google Scholar) and for the development of a reference map of phosphoproteins. A reference gel is defined as 0.2 mg of total protein from each of the five developmental stages pooled and resolved by 2-DGE. SDS-PAGE was performed by standard methods utilizing 4% T, 2.6% C stacking gels, pH 6.8, and 12% T, 2.6% C separating gels, pH 8.8 (43Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). The % T is the total monomer concentration expressed in grams/100 ml, and % C is the percentage of cross-linker. The stacking and separating gel buffer concentrations were 0.125 m Tris-HCl, pH 6.8, and 0.375 m Tris-HCl, pH 8.8, respectively. The reservoir buffer concentration was 0.025 m Tris, 0.192 m glycine, pH 8.3. All gel and reservoir buffers contained SDS to a final concentration of 0.1% (w/v). SigmaMarker or protein samples were heated for 5 min at 75 °C in SDS loading buffer (5% (v/v) glycerol, 60 mm SDS, 100 mm DTT, 0.03 mm bromphenol blue, and 60 mm Tris-HCl, pH 6.8) and were cooled to room temperature before loading to 10 and 15% gels. A total of 500 μg was loaded per lane, and electrophoresis was conducted overnight in a Hoefer vertical SE600 electrophoresis unit (GE Healthcare catalog number 80-6171-96) at room temperature until the dye reached the bottom of the gel. Gels were stained with colloidal Coomassie Brilliant Blue (CBB) to detect protein. For phosphoprotein detection, 2-D gels were stained with a modified protocol using Pro-Q DPS (44Agrawal G.K. Thelen J.J. Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins.Proteomics. 2005; 5: 4684-4688Crossref PubMed Scopus (90) Google Scholar). Briefly all gels were treated with fixation solution (2 × 30 min), washed with deionized water (2 × 15 min), stained with 3-fold diluted Pro-Q DPS in deionized water (120 min), destained with destaining solution (4 × 30 min) to remove gel-bound nonspecific Pro-Q DPS, and washed again with deionized water (2 × 5 min). Following scanning of Pro-Q DPS-stained gels, the same gels were then overstained with colloidal CBB G-250 to detect proteins (45Mooney B.P. Thelen J.J. High-throughput peptide mass fingerprinting of soybean seed proteins: automated workflow and utility of UniGene expressed sequence tag databases for protein identification.Phytochemistry. 2004; 65: 1733-1744Crossref PubMed Scopus (124) Google Scholar). All gels in the incubation solution were constantly shaken on an orbital shaker (GeneMate, ISC Bioexpress) at room temperature at speeds of 35 rpm. Following the Pro-Q DPS procedure, gels were imaged using an FLA 5000 laser scanner (Fuji Medical Systems, Stamford, CT) with 532 nm excitation and 550-nm bandpass emission filter. Data were collected as 100-μm resolution, 16-bit TIFF files using the Image Gauge Analysis software (Fuji Medical Systems). With this software, fluorescent protein signals in 2-D gels were displayed as dark spots. To quantify phosphoprotein spots in profile mode, 2-D gels were analyzed using ImageMaster 2D Platinum software version 5 (hereafter called ImageMaster software; GE Healthcare) as described by Hajduch et al. (20Hajduch M. Ganapathy A. Stein J.W. Thelen J.J. A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database.Plant Physiol. 2005; 137: 1397-1419Crossref PubMed Scopus (311) Google Scholar). Under applied stringent criteria to select phosphoprotein spots for quantification and expression profiling, only those spots were analyzed that were present in all four gels derived from independent biological samples of one developmental stage and expressed at least in two of five developmental stages. The relative volumes of high quality phosphoprotein spots were determined followed by establishment of their expression profiles. To obtain the statistical significance of the variation of each expressed phos