Proteomic Analysis of Seed Filling in Brassica napus. Developmental Characterization of Metabolic Isozymes Using High-Resolution Two-Dimensional Gel Electrophoresis

Abstract

Brassica napus (cultivar Reston) seed proteins were analyzed at 2, 3, 4, 5, and 6 weeks after flowering in biological quadruplicate using two-dimensional gel electrophoresis. Developmental expression profiles for 794 protein spot groups were established and hierarchical cluster analysis revealed 12 different expression trends. Tryptic peptides from each spot group were analyzed in duplicate using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and liquid chromatographytandem mass spectrometry. The identity of 517 spot groups was determined, representing 289 nonredundant proteins. These proteins were classified into 14 functional categories based upon the Arabidopsis (Arabidopsis thaliana) genome classification scheme. Energy and metabolism related proteins were highly represented in developing seed, accounting for 24.3% and 16.8% of the total proteins, respectively. Analysis of subclasses within the metabolism group revealed coordinated expression during seed filling. The influence of prominently expressed seed storage proteins on relative quantification data is discussed and an in silico subtraction method is presented. The preponderance of energy and metabolic proteins detected in this study provides an in-depth proteomic view on carbon assimilation in B. napus seed. These data suggest that sugar mobilization from glucose to coenzyme A and its acyl derivative is a collaboration between the cytosol and plastids and that temporal control of enzymes and pathways extends beyond transcription. This study provides a systematic analysis of metabolic processes operating in developing B. napus seed from the perspective of protein expression. Data generated from this study have been deposited into a web database (http://oilseedproteomics.missouri.edu) that is accessible to the public domain.Brassica napus (also known as rape and oilseed rape) is the third largest oilseed crop in the world, providing approximately 13% of the world's supply of vegetable oil. B. napus seeds produce oil and protein as the main storage compounds out of the three principal storage reserves (proteins, oil [triacylglycerols], and carbohydrates [starch]) found in plant seeds (Norton and Harris, 1975;Murphy et al., 1989;King et al., 1997;Schwender and Ohlrogge, 2002). These compounds support early seedling growth, and in nature the relative proportions of these compounds vary dramatically among different plant seeds. Seeds of legume species such as soybean (Glycine max) and pea (Pisum sativum) produce seeds with 20% to as much as 40% protein and 20% oil, while B. napus produces seeds with approximately 40% oil and 15% protein (Gunstone et al., 1995).Biochemical and molecular studies are beginning to define the biosynthetic pathways responsible for accumulation of these storage components in B. napus

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

“…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. Spot intensities are surrogates for the concentration of the proteins and peptides in the experimental samples.…”

“…Analysis of LC-MS/MS data was performed as described earlier. 24 Briefly, database searches were performed on a local licensed copy of Sequest 28,29 as part of the BioWorks 3.1SR1 software suite (ThermoFisher, San Jose, CA). Search parameters were set as follows: enzyme, trypsin; number of internal cleavage sites, 2; mass range, 400-1600; threshold, 500; minimum ion count, 35; peptide mass tolerance, 1.50 Da; variable modifications, oxidation (M); static modification, carboxyamidomethylation (C).…”

“…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.…”