In-Depth Investigation of the Soybean Seed-Filling Proteome and Comparison with a Parallel Study of Rapeseed  

Abstract

To better understand the metabolic processes of seed filling in soybean (Glycine max), two complementary proteomic approaches, two-dimensional gel electrophoresis (2-DGE) and semicontinuous multidimensional protein identification technology (Sec-MudPIT) coupled with liquid chromatography-mass spectrometry, were employed to analyze whole seed proteins at five developmental stages. 2-DGE and Sec-MudPIT analyses collectively identified 478 nonredundant proteins with only 70 proteins common to both datasets. 2-DGE data revealed that 38% of identified proteins were represented by multiple 2-DGE species. Identified proteins belonged to 13 (2-DGE) and 15 (Sec-MudPIT) functional classes. Proteins involved in metabolism, protein destination and storage, and energy were highly represented, collectively accounting for 61.1% (2-DGE) and 42.2% (Sec-MudPIT) of total identified proteins. Membrane proteins, based upon transmembrane predictions, were 3-fold more prominent in Sec-MudPIT than 2-DGE. Data were integrated into an existing soybean proteome database (www.oilseedproteomics.missouri.edu). The integrated quantitative soybean database was compared to a parallel study of rapeseed (Brassica napus) to further understand the regulation of intermediary metabolism in protein-rich versus oil-rich seeds. Comparative analyses revealed (1) up to 3-fold higher expression of fatty acid biosynthetic proteins during seed filling in rapeseed compared to soybean; and (2) approximately a 48% higher number of protein species and a net 80% higher protein abundance for carbon assimilatory and glycolytic pathways leading to fatty acid synthesis in rapeseed versus soybean. Increased expression of glycolytic and fatty acid biosynthetic proteins in rapeseed compared to soybean suggests that a possible mechanistic basis for higher oil in rapeseed involves the concerted commitment of hexoses to glycolysis and eventual de novo fatty acid synthesis pathways.Plant seeds accumulate proteins, oils, and carbohydrates because these nitrogen and carbon reserves are necessary for early seed germination and seedling growth (for review, see Weber et al., 2005). These reserve components are synthesized during an extended phase of seed development, loosely termed seed filling. Seed filling is the period when rapid metabolic and morphological (size, weight, and color) changes occur, encompassing cellular processes that include cell expansion and the early stage of desiccation (Rubel et al., 1972;Mienke et al., 1981;Agrawal and Thelen, 2006). Seed filling is also the period that largely determines the relative levels of storage reserves in seeds. The relative proportion of storage components in seeds varies dramatically among different plant species. For example, soybean (Glycine max) seed contains approximately 40% protein and 20% oil (Hill and Breidenbach, 1974;Ohlrogge and Kuo, 1984). In contrast, seed of oilseed rape (Brassica napus; also called rapeseed or canola) contains approximately 15% protein and 40% oil (Norton and Harris, 1975). To gain insight into...

The Proteome of Seed Development in the Model Legume Lotus japonicus

“…So far, 746 unique gene accession numbers identified from green and mature seeds constitute the Lotus resource. Similar 2-D gel or Sec-MudPIT-based proteomic studies of seed filling in Medicago and soybean identified 226 and 647 unique proteins, respectively (Gallardo et al, 2003(Gallardo et al, , 2007Hajduch et al, 2005;Agrawal et al, 2008). Comparing these Medicago and soybean proteins against the identified Lotus seed proteins (BLASTP e # 1 3 10 26 ), 39% of the proteins from Lotus were also identified in Medicago, and with soybean the overlap was 60%.…”

“…Refining the bioinformatics-based comparison presented above, we compiled starch enzyme identifications from the five seed proteomic studies ( Fig. 7; Gallardo et al, 2003Gallardo et al, , 2007Hajduch et al, 2005;Agrawal et al, 2008). Although legume seed starch metabolism may be different from the well-studied processes in leaves and in cereal grains, this approach provided good coverage of known starch biosynthetic and degradation pathways (Fig.…”

Post-Genomics Studies of Developmental Processes in Legume Seeds

“…More recently, techniques of shotgun protein sequencing have developed which, with appropriate informatics support, can provide a much more comprehensive picture of the proteome (Malmstrom et al, 2007). These analyses have established reference maps of the protein complement of developing or mature seed in model and crop species, allowing for the identification of species-specific features (Agrawal et al, 2008;Gallardo et al, 2008). The methods have been applied to total protein extracts, or in some cases to subcellular fractions, as for example in the analysis of nuclear proteins (Repetto et al, 2008).…”

“…For the entire seed, the citations refer to post-genomics experiments carried out in various legume species at the level of the transcriptome (Firnhaber et al, 2005;Buitink et al, 2006;Gallardo et al, 2007;Benedito et al, 2008;Verdier et al, 2008), metabolome (Vigeolas et al, 2008), ionome (Sankaran et al, 2009), and proteome. At the level of the proteome, some citations refer to the mature seed (Watson et al, 2003;Magni et al, 2007;Bourgeois et al, 2009;Krishnan et al, 2009;Natarajan et al, 2009), to the kinetics of seed development (Hajduch et al, 2005;Gallardo et al, 2007;Agrawal et al, 2008;Dam et al, 2009), and to a functional study for identifying targets of thioredoxin (Alkhalfioui et al, 2007). For the separated seed tissues, the citations refer to transcriptome and proteome analyses in soybean (Le et al, 2007) and M. truncatula (Boudet et al, 2006;Zhang et al, 2006;Gallardo et al, 2007).…”

Phosphoproteomic Analysis of Seed Maturation in Arabidopsis, Rapeseed, and Soybean

“…A better understanding of the biology and biochemistry of seed development can provide opportunities for the rational engineering of seed content. In this regard, the recent use of transcriptomics and proteomics approaches has begun to provide global views of the compendium of regulatory, structural, and enzymatic proteins involved in carbon assimilation and reserve deposition in the diversity of model and agronomic oilseeds (Ruuska et al, 2004;Hajduch et al, 2005;Katavic et al, 2006;Agrawal et al, 2008;Houston et al, 2009). Although the metabolic pathways involved in seed maturation have been largely predicted using both "omics" (Hajduch et al, 2010) and in vivo labeling approaches (Schwender et al, 2003; for review, see Baud and Lepiniec, 2009), the regulation of the activities in these pathways is only beginning to be discovered.…”