A systematic proteomic study of seed filling in soybean. Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database

Abstract

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.

Figure 1.

Development of soybean seeds during the experimental period. A, Whole seeds at the five stages of seed filling. Experimental sampling began exactly 2 WAF and continued at precisely 7-d intervals until 6 WAF. B, Individual seed size characteristics, including length (x), thickness (y), and width (z), were determined using an ultramicrometer. Each value is the average of 10 seeds; sd is denoted by error bars. C, Seed fresh and dry mass during the experimental period expressed as mass per seed. Values are the average of 10 determinations; sd is shown. D, Total protein content per seed during the investigated period of seed filling. Values are the average of 10 determinations; sd is shown.

Figure 2.

Analysis of proteins (1 mg) isolated from immature soybean seeds of 2-, 3-, 4-, 5-, and 6-WAF periods by 2-DE in combination with colloidal Coomassie Brilliant Blue staining. A, Protein analysis using wide-range IPG strips with pH range from 3 to 10. B, Two-dimensional electrophoresis of highly dense region of pH 4 to 7 using narrow-range IPG strips. Isoelectric points (pI) and molecular mass (in kD) are noted. Reference gel is a composite of all 5 seed stages obtained by pooling 0.2 mg of protein from each stage.

Figure 3.

Experimental design for protein expression analysis. Four biological replicates for each developmental stage were analyzed using 2-DE. For quantification, only high-quality protein spots that were present in at least three out of the four gels were analyzed. Expression analyses were carried out using ImageMaster 2-D Platinum software version 5.0. Spots from analyzed gels were matched with spots from reference (pooled) gel and relative volumes (RV) were calculated for each spot group. Average relative volume for matched spots from replicate gels was calculated, and data were plotted onto a line graph. Finally expression profile data were uploaded onto a Web-based data repository (http://oilseedproteomics.missouri.edu/).

Figure 4.

Experimental design for protein identification analyses using MALDI-TOF-MS. Protein spots with expression profiles were excised from reference (pooled) gels from pH ranges 3 to 10 and 4 to 7. For the pH 3 to 10 gel, spots were excised only from the pH 7 to 10 region to minimize redundancy. Each spot was trypsin digested, and peptides were analyses using MALDI-TOF-MS. Resultant spectra were searched against the NCBI soybean UniGene database using the MS-Fit program of Protein Prospector. All 422 identified proteins are listed in Table I and can also be accessed from a Web database.

Figure 5.

The functional distribution of nonredundant proteins identified from developing soybean seed. From 422 total identified proteins, 216 had nonredundant functions. The pie chart shows the distribution of these nonredundant genes after functional classification. Functional classification was based upon nomenclature by Bevan et al. (1998).

Figure 6.

Composite protein expression profiles of gene function categories. The combined expression profiles were calculated as the sum of all relative volumes for each protein in the functional category. A, The combined expression profile of all 82 proteins involved in metabolism (left). Additional plot (right) shows 12 proteins involved in metabolism of amino acids (01.01), 17 in nitrogen and sulfur (01.02), 24 proteins involved in metabolism of sugars and polysaccharides (01.05), and 27 that are involved in metabolism of lipids and sterols (01.06). B, Composite expression profiles of 80 proteins involved in protein destination and storage (left). Additional plot (right) is for 12 proteins involved in folding and stability (06.01), 14 involved in proteolysis (06.13), and for 54 storage proteins (06.20). C, The combined expression profile of 45 proteins identified as transporters. More detailed view is shown by 7 ion transporters (07.01), 23 sugar transporters (07.07), and 15 ABC-type transporters (07.25). D, The combined expression profile of 27 proteins involved in disease and defense mechanisms. Additional charts show 3 resistance proteins (11.01), 4 stress responses proteins (11.05), and 20 detoxification proteins (11.06).

Figure 7.

Composite expression profiles of various classes of proteins during soybean seed filling. The two principal soybean seed storage proteins, glycinin and β-conglycinin, both were represented by 26 spots. The figure also shows additional classes: 14 energy proteins, 45 cell growth and division proteins, 8 proteins involved in protein synthesis, 20 signal transduction proteins, and 12 secondary metabolism proteins.

Figure 8.

Expression profiles of 92 proteins annotated in the UniGene database as unknown. The expression profiles were grouped into five expression profile groups, and the percentage of unknown proteins in each group was calculated.