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Plant Physiology 137:1397-1419 (2005) © 2005 American Society of Plant Biologists A Systematic Proteomic Study of Seed Filling in Soybean. Establishment of High-Resolution Two-Dimensional Reference Maps, Expression Profiles, and an Interactive Proteome Database1,[w]Department of Biochemistry, Life Sciences Center (M.H., J.J.T.), and Computer Science Department (A.G., J.W.S.), University of Missouri, Columbia, Missouri 65211
A high-throughput proteomic approach was employed to determine the expression profile and identity of hundreds of proteins during seed filling in soybean (Glycine max) cv Maverick. Soybean seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This led to the establishment of high-resolution proteome reference maps, expression profiles of 679 spots, and corresponding matrix-assisted laser desorption ionization time-of-flight mass spectrometry spectra for each spot. Database searching with these spectra resulted in the identification of 422 proteins representing 216 nonredundant proteins. These proteins were classified into 14 major functional categories. Proteins involved in metabolism, protein destination and storage, metabolite transport, and disease/defense were the most abundant. For each functional category, a composite expression profile is presented to gain insight into legume seed physiology and the general regulation of proteins associated with each functional class. Using this approach, an overall decrease in metabolism-related proteins versus an increase in proteins associated with destination and storage was observed during seed filling. The accumulation of unknown proteins, sucrose transport and cleavage enzymes, cysteine and methionine biosynthesis enzymes, 14-3-3-like proteins, lipoxygenases, storage proteins, and allergenic proteins during seed filling is also discussed. A user-intuitive database (http://oilseedproteomics.missouri.edu) was developed to access these data for soybean and other oilseeds currently being investigated.
Soybeans are responsible for approximately $12 billion in annual crop value and more than $5 billion in annual export value to the U.S. economy (Gunstone, 2001  ). Soybeans (and other oilseeds) are a significant source of fatty acids and proteins for human and animal nutrition as well as for nonedible uses, including industrial feedstocks and combustible fuel (for review, see Thelen and Ohlrogge, 2002). The major source of these commodities is the seed, and a better understanding of this structure will be important for future biotechnology efforts.
During a 4- to 5-week period of soybean seed development, most of the storage reserves for germination are synthesized; this phase of development is generally termed seed filling. At maturation, approximately 41% of soybean seed dry weight is storage protein (Hill and Breidenbach, 1974
Seed filling has been investigated recently in Arabidopsis (Arabidopsis thaliana) on a global, transcriptome level. Microarray analyses of developing seeds revealed complex networks of gene expression (Girke et al., 2000
Other proteomics investigations of legume seeds include two surveys of the principal proteins expressed in mature soybean seeds (Herman et al., 2003
Staging and Characterization of Developing Soybean Seed
The primary objective of this study was to characterize global protein expression during the seed-filling phase of soybean seed development. For the best coverage of this period, whole seeds were analyzed at precisely 2, 3, 4, 5, and 6 weeks after flowering (WAF). The experimental period included the late morphogenic phase (2 WAF), which is completed when seeds are about 2 mm long, the period of cell division (3, 4 WAF), and the cell enlargement period (5, 6 WAF) but not the early embryogenesis or the seed maturation phases (Mienke et al., 1981
Narrow-Range Isoelectric Focusing Is Necessary for High-Resolution Proteome Maps Whole proteins from staged developing soybean seed were resolved and detected using high-resolution two-dimensional electrophoresis (2-DE) followed by colloidal Coomassie Blue staining. Initial analyses were performed with immobilized pH gradient (IPG) strips that ranged from pH 3 to 10 (Fig. 2A). It was observed that the region from pH 4 to 7 was a highly dense area on the proteome map; therefore, additional analyses with pH 4 to 7 IPG strips were performed to improve spot resolution (Fig. 2B). The 2-DE maps showed a highly dynamic proteome during soybean seed development. The late morphogenesis phase of seed development (2 WAF) and the early cell division stage (3 WAF) showed similar 2-DE spot patterns. Late cell division (4 WAF) and cell enlargement (5 and 6 WAF) periods were characterized by an increasing abundance of seed storage proteins, which accounted for approximately 35%, 53%, and 60% of total seed protein, respectively.
Altogether 679 Spot Groups Were Quantified Using 2-DE After 2-DE, gels were imaged and analyzed using ImageMaster Platinum software (Amersham Biosciences, Piscataway, NJ). For protein expression analyses, the volume of each spot was expressed as relative volume, a ratio of individual spot volume to the sum of spot volumes for all analyzed spots. Analysis of relative volume, instead of raw volumes, corrected for subtle experimental variations due to protein loading and staining. Moreover, the relative volumes were adjusted with correction constants to enable direct comparison between the pI 4 to 7 and pI 3 to 10 datasets. The relative volumes for a particular spot, obtained from at least three biological analyses, were averaged. Within these protein groups, the protein expression data and SD values for each developmental stage were plotted on a line graph (Fig. 3).
To ensure the quality of expression data, threshold criteria were established for image analysis. To be included in expression analyses, each spot group needed to be represented in at least three out of four biological replicates. In total, 488 proteins satisfied these criteria from 2-DE gels of pH range 4 to 7. An additional 191 proteins from pH 3 to 10 gels (pH 7–10 region only) also satisfied these criteria (Fig. 4). The software used for this analysis assigned a unique number to each detected spot within each gel in the series (spot 3514 in Fig. 3). After analyzed gels were matched to a reference gel, protein groups were established and assigned a spot group number (1100 in Fig. 3) corresponding to the number of that particular spot on the reference gel. Using this systematic approach, the expression profiles for 679 spot groups were obtained in biological triplicate for each of the five developmental stages.
Peptide Mass Fingerprinting Using the Soybean UniGene Database Yielded 422 Protein Assignments
Each of the 679 proteins with confirmed expression profiles was excised from reference gels for identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS; Fig. 4). After MALDI-TOF analysis of tryptic peptides, mass tags were searched against the soybean UniGene database using the MS-FIT program of Protein Prospector (University of California, San Francisco, CA). The identification output was a list of 50 possible identifications (candidates) sorted according to their Molecular Weight Search (MOWSE) scores (Pappin et al., 1993
Each of the 422 identified proteins was classified into one of 15 functional categories as established by Bevan et al. (1998
A database, constructed and appended for archiving the data from this investigation, was the Web portal for proteomics research on oilseed plants (http://oilseedproteomics.missouri.edu/). This Web database offers two result viewing options: (1) a list of identified proteins sorted into plant functional categories with identification numbers linked to expression profile and identification data; and (2) interactive images of pooled gels pH 4 to 7 and pH 3 to 10 with active spot links that will lead users to expression and identification data. The protein identification data contain basic parameters describing PMF data and also a list of masses and intensities of tryptic peptides to allow for independent validation and updating of protein assignments.
Seed development can be simplified by categorizing this complex process into three sequential, temporal phases: embryogenesis, seed filling, and maturation. The seed-filling phase of development is the longest phase and is a period of cell division and cell expansion as well as storage product synthesis (Mienke et al., 1981 ). The metabolic events that occur during seed filling determine the overall storage composition of seed, and targeted transgenic alteration of these pathways can have a major impact on seed quality traits (Thelen and Ohlrogge, 2002). Despite its importance in quantitative trait determination, the seed-filling phase of oilseed development is poorly understood from a regulatory perspective. A recent investigation on the proteomics of seed filling in M. truncatula has been reported (Gallardo et al., 2003). The authors profiled and identified 84 proteins at various stages of M. truncatula seed development, and 23 of those identified proteins were storage proteins specific for Medicago. Interestingly, only 15 proteins from this study on soybean seed filling also were observed in Medicago, a related legume. Many of those common proteins, including Suc synthase and Met synthase, shared similar developmental expression profiles. Recently, two studies were published on proteomics of mature soybean seeds; Herman et al. (2003) identified 111 proteins, and Mooney and Thelen (2004) identified 44 proteins. When compared with this study, 55 and 22 proteins, respectively, were also identified and characterized during the seed-filling period of the current investigation.
To characterize global expression trends of proteins involved in different processes, we established composite expression profiles by summing protein abundance, expressed as relative volume, for each protein in each functional class for the five seed stages (Figs. 6 and 7). Relative abundances of metabolic proteins decreased during the experimental period (Fig. 6A), suggesting metabolic activity curtails as seeds approach maturity. Interestingly, proteins involved in lipid and sterol metabolism decreased from 2 to 4 WAF, but after 4 WAF their abundance slightly increased. The protein destination and storage class of proteins increased during late seed filling, and this was due to the preponderance of seed storage proteins (Figs. 6B and 7). The transporter class of proteins, which includes the ubiquitous Suc-binding proteins, exhibited almost constant but slightly increased expression during the experimental period (Fig. 6C). Disease- and defense-related proteins were highly abundant at the early stage of seed filling; later their abundance decreased to about 50% and from 4 WAF was stable. Proteins involved in energy production increased in abundance during seed filling (Fig. 7), whereas cell growth and division proteins as well as signal transduction proteins each had decreasing expression profiles. Proteins involved in protein synthesis and secondary metabolism decreased in abundance during the experimental period.
Unknown Proteins Account for 22% of All Identified Proteins Out of 422 identified proteins, 92 were annotated as unknown. The classification of unknown proteins based on their expression profiles may provide clues about possible functions in seed development (Fig. 8). Based upon the similarities between expression profiles of unknown proteins and expression profiles for each major functional class (Figs. 6 and 7), it is possible to speculate on the function of these unknown proteins. Often expression of different isoforms of the same protein have different trends (i.e. Suc-binding proteins discussed in the next paragraph; 7 increased, 5 decreased). For this reason, the composite expression profile of the functional class taken for comparison needs to contain a significant number of proteins. Taking into account the number of identified proteins in this study, functional classes with 40 and more proteins were considered suitable for such analysis.
The largest group of unknown proteins (57%) decreased in expression during seed development. This expression profile is characteristic for proteins involved in metabolism (Fig. 6A) and cell growth/division (Fig. 7). Thirteen percent of unknown proteins increased in abundance during seed filling, and this was characteristic for proteins associated with protein destination and storage (Fig. 6B). Eight percent of unknown proteins had stable relative abundance through the experimental period, similar to the transporter class of proteins (Fig. 6C). For the remaining 15% that had maximum abundance and 7% that had minimal abundance at 4 WAF, it is not possible to correlate function because no functional class shares these expression profiles.
To provide carbon for all major classes of storage products, developing seeds import Suc, which is cleaved by Suc synthase (Gifford and Evans, 1981
In legumes, it was reported that the transcript level of Suc synthase steadily increased until the middle of development and declined thereafter. Suc levels approximately paralleled levels of Suc synthase mRNA (Heim et al., 1993
The products of Suc synthase are largely metabolized through the glycolytic pathway, before utilization in subsequent reactions, such as amino acid and fatty acid biosynthesis. Oilseeds have a complete set of glycolytic enzymes in plastids, in addition to the cytosolic pathway (Miernyk and Dennis, 1983
Figure 6A gives a general overview on the expression of 82 identified proteins associated with metabolism. The expression of metabolic proteins gradually decreased during seed filling. It is well known that soybean seeds and seed proteins are deficient in the sulfur-containing amino acids Cys and Met, and a major research effort is concentrated on increasing sulfur-containing amino acids within soybean seeds. The key enzymes for biosynthesis of both amino acids, Cys synthase (spot 984) and Met synthase (spot 281), were found in this study. The peak of expression for Cys synthase was at 3 WAF, after which the expression level decreased rapidly. In the case of Met synthase, the relative abundance rapidly decreased through seed filling and was about 5 times lower at 6 WAF when compared with 2 WAF. This is in agreement with previous studies in which Met synthase was reported to be present at low levels in mature Arabidopsis seeds (Gallardo et al., 2002
The 14-3-3 proteins play important roles in various aspects of plant and metabolic processes (for review, see Roberts, 2003
Lipoxygenase (LOX) activities have been shown to be induced during plant defense responses (Keppler and Novacky, 1987
In this study, we identified 8 unique LOX representing 16 individual spots. Five spots (204, 746, 758, 982, and 2373) were identified as LOX L9 originally isolated from soybean var Paldal (Ahn et al., 2003
The two major seed storage proteins from soybean are multimeric with Svedberg coefficients of 7 and 11S. The 7S globulins are comprised of
Three soybean proteins, Gly m Bd 60 K, Gly m Bd 30 K, and Gly m Bd 28 K, represent the main seed allergens in soybean-sensitive patients (Ogawa et al., 2000
Soybean Kunitz trypsin inhibitor (SKTI) is a small monomeric, nonglycosylated protein containing 181 residues that has been characterized as a human food allergen (Burks et al., 1994
Plant Material and Growth Conditions
Soybean plants (Glycine max L. Maverick) were grown (ProMix soil, Quakertown, PA) in a greenhouse in Columbia, Missouri, with supplemental lighting (16-h-light/8-h-dark cycle, 26°C day/21°C night). Plants were not nodulated and were fertilized at 2-week intervals (all-purpose fertilizer, 15-30-15). Soybean flowers were tagged after opening (between 1 and 3 PM CST), and developing seed were collected precisely 2, 3, 4, 5, and 6 WAF, i.e. 14, 21, 28, 35, and 42 DAF. Seed length, thickness, and width were measured using a micrometer. Fresh and dry weight as well as total protein content were measured at each seed stage. Protein was quantified from whole-seed homogenates using a dye-binding protein assay (Bradford, 1976
Total protein was isolated from developing seed according to a modified phenol-based procedure based upon that of Hurkman and Tanaka (1986)
Proteins pellets were resuspended in 1 mL of IEF sample extraction media (8 M urea, 2 M thiourea, 2% [w/v] CHAPS, 2% [v/v] Triton X-100, 50 mM dithiothreitol) by vortexing at low speed for 1 h. Insoluble matter was removed by centrifugation for 20 min at 14,000g. Protein concentration was determined using a protein assay from Bio-Rad (Hercules, CA), based upon the modified procedure of Bradford (1976) Following IEF, IPG strips were removed from focusing tray, blotted to remove mineral oil, and incubated in SDS equilibration buffer (1.5 M Tris-HCl, 6 M urea, 30% [v/v] glycerol, 5% [w/v] SDS) for 15 min with 2% (w/v) dithiothreitol followed with another 15 min with 2.5% (w/v) of iodoacetamide. IPG strips were then rinsed with SDS running buffer (25 mM Tris, 0.192 M Gly, and 0.1% [w/v] SDS) and placed onto 12% acrylamide gels. Strips were then overlayed with 0.5% (w/v) agarose in SDS running buffer. Second-dimension SDS-PAGE was performed in an Ettan Dalt 12 electrophoresis unit (Amersham Biosciences, Piscataway, NJ) for 2 W/gel for 16 h or until dye migrated off the gel. Following SDS-PAGE, gels were washed in deionized water three times for 15 min and stained for a least 12 h with colloidal Coomassie (20% [v/v] ethanol, 1.6% [v/v] phosphoric acid, 8% [w/v] ammonium sulfate, 0.08% [w/v] Coomassie Brilliant Blue G-250).
Image acquisition was performed using a ScanMaker 9800XL (Microtek, Carson, CA) with a resolution of 300 dpi and 16-bit grayscale pixel depth. Image analysis was carried out with ImageMaster 2D Platinum software version 5.0 (Amersham Biosciences, Uppsala), which allows spot detection, quantification, background subtraction, and spot matching among multiple gels. Each stage of seed development was analyzed at a minimum in biological triplicate. Protein spots were selected for profile analysis only if they were confirmed in at least three independent sample sets. For the purpose of spot analysis, a reference or pooled gel was created by pooling equal amounts of protein (200 µg) of each seed stage onto one gel. To compensate for subtle differences in sample loading, gel staining, and destaining, the volume of each spot (i.e. spot abundance) was normalized as relative volume. This normalization method provided by ImageMaster 2D Platinum software divides each spot volume value by the sum of total spot volume values to obtain individual relative spot volumes. Total spot volume refers to the sum volume of all spots chosen for analysis; therefore, the volumes of spots excluded from the analysis were not considered. After spot detection, quantification, and background subtraction, each analyzed gel was matched individually to the reference gel, and matched spots were grouped into subclasses.
To enable direct comparison between protein relative abundances (expressed in relative volumes) from the two independent datasets (i.e. pI 4–7 and pI 3–10), a second normalization was performed. For each experimental stage, the total spot volume was calculated for each gel in the analysis separately, and the correction constants for gels pI 4 to 7 (CpI 4-7) and pI 3 to 10 (CpI 3-10) were calculated using following formulas:
Each protein spot from a pooled reference gel was arrayed into 96-well MultiScreen plate model R5.5µm hydrophilic PTFE membrane, glass-filled polypropylene plates (Millipore, Bedford, MA) using 3.0-mm-diameter pins on the GelPix robotic spot excision station (Genetix, Milton, UK) or using a 1.5-mm-diameter manual excision pen (Gel Company, San Francisco). After arraying of protein spots, gel plugs were destained three times in 50% acetonitrile, 50 mM ammonium bicarbonate for 15 min at room temperature with gentle agitation using a microplate shaker. Destaining solution was evacuated from the bottom of the filter plates using a vacuum manifold (Millipore). Gel plugs were dehydrated in 100% acetonitrile for 5 min at room temperature. Acetonitrile was evacuated, and plates were gently blotted to filter paper to remove residual acetonitrile. Then 96-well polypropylene V-bottom sample collection plates were placed underneath each MultiScreen plate. The gel plugs were digested with 50 µL of sequencing grade trypsin manufactured by Promega (Madison, WI; 0.004 µg/µL in 50 mM ammonium bicarbonate) for 16 h at 37°C. After digestion, tryptic peptides were extracted from gel plugs using 100 µL of extraction solution (60% [v/v] acetonitrile, 1% [v/v] formic acid). After 10 min of extraction with gentle agitation, peptides were collected into V-bottom 96 collection plates by centrifugation at 2,000g for 2 min. The extraction procedure was repeated with the same amount of extraction solution. Extracted tryptic peptides were dried by centrifugal vacuum evaporation and stored at –80°C until analysis.
Prior to mass spectrometry, peptides were desalted using C18 microbed chromatography (ZipTip; Millipore). Dried tryptic peptides were resuspended in 10 µL of 1% formic acid. ZipTips were conditioned in 100% acetonitrile, followed by 70% (v/v) acetonitrile, 1% (v/v) formic acid and finally in 1% (v/v) formic acid. After peptide binding and washing with 1% (v/v) formic acid, peptides were eluted from C18 matrix with 7 µL of elution solution (70% [v/v] acetonitrile, 1% [v/v] formic acid). Then 0.5 µL of eluted peptides was applied to a 96 x 2 Teflon MALDI plate and mixed on target with 0.5 µL of matrix solution: 10 mg/mL
Programming for the Web database was scripted through C (Kerninghan and Richie, 1978
A proteomics database archiving all protein expression and identification data discussed here can be found at http://oilseedproteomics.missouri.edu/. The supplemental files online contain all expression profile data.
We thank Alan Marshall for database maintenance and server space. We also thank Professor Dong Xu (University of Missouri, Columbia) for providing computational resources for script writing and data processing. Received November 16, 2004; returned for revision December 29, 2004; accepted December 30, 2004.
1 This work was supported by the National Science Foundation Plant Genome Research Program (Young Investigator Award no. DBI–0332418). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.056614. * Corresponding author; e-mail thelenj{at}missouri.edu; fax 573–884–9676.
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ABSTRACT
RESULTS
-conglycinin (Roberts and Briggs, 1965
and 
-globulin. The desired amount of protein (1 mg) was added to a 1.5-mL tube, and volume was brought up to 450 µL with IEF extraction media. Ampholytes (2.25 µL) were added, mixed by vortexing, and centrifuged for 5 min at 14,000g to remove the remaining insoluble matter. The protein mix was transferred into an IEF tray, and an IPG (24 cm; Amersham Biosciences, Uppsala) was carefully placed onto the protein sample and allowed to rehydrate for 1 h prior to the addition of mineral oil. The IEF tray was placed into protean IEF cell unit (Bio-Rad). Active rehydration (10 h at 50 V) was performed, and this was directly linked to the three-step focusing protocol: 100 V for 100 Vh, 500 V for 500 Vh, and 8,000 V for 99 kVh. 