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.

Proteomic Analysis of Near-Isogenic Sunflower Varieties Differing in Seed Oil Traits

“…The differentially expressed protein spots (FDR e10%) were excised from corresponding preparative CBB-stained 2-DE gel and trypsin-digested as described previously. 23 Liquid chromatography-tandem mass spectrometry using a ProteomeX LTQ ion trap (Thermo-Fisher, San Jose, CA) was performed according to manufacturer's instructions for high-throughput protein identification as described previously. 24 Statistical Analysis.…”

“…Total protein was isolated from developing seed and subjected to two-dimensional protein electrophoresis (2-DE) as described previously. 23 Protein quantification was performed in triplicate using the Coomassie dye binding assay (Bio-Rad, Hercules, CA) against standard curve of chicken γ-globulin.…”

“…This is based upon previous proteomic studies of seed filling in soybean and rapeseed that revealed enzymes involved in sucrose assimilation, glycolysis, and de novo fatty acid synthesis are all prominent at this period of seed development. 23,24 Because oil content is a complex trait possibly involving multiple intermediary metabolic pathways in addition to de novo fatty acid synthesis, we expected to observe more protein expression differences between the RHA280 and 801 lines than a parallel analysis of inbred lines varying in oleic acid composition, which is largely determined by a single desaturase activity, FAD2. 7 Indeed, 77 protein spots were differentially expressed in the oil content inbred lines versus 42 differential proteins for the two inbred lines varying in oleic acid composition.…”

“…Glycolytic enzymes fructokinase (spots 357, 362) and plastid phosphoglycerate kinase (PGA kinase, spot 406) were slightly higher in RHA801, whereas enolase 2 (spot 139) was strongly upregulated in the high oil line (Figure 4). Previous proteomic analysis of seed filling in soybean 23 and rapeseed 24 indicated high expression levels of sucrose synthase (SuSy) but not invertase, suggesting that sucrose assimilation proceeds principally through SuSy. Because fructose is one of the products of SuSy (UDP-glucose is the other), it is possible that fructokinase, in collaboration with SuSy, could play an important role in phloem unloading during seed filling in sunflower.…”

“…Interestingly, tomato fructokinase has previously been shown to have unusual regulatory properties and may play a major role in sink strength of developing fruit. 36,37 Because phosphoenolpyruvate (PEP) is the likely precursor for plastid de novo fatty acid synthesis during seed development, 23,24,38 enolase could be important in "pulling" glycolyticand RuBisCO-generated 3-phosphoglycerate (3-PGA) into PEP and, therefore, de novo fatty acid synthesis. This model would presume that 3-PGA and 2-PGA are in equilibrium through phosphoglycerate mutase.…”