Abstract
Seed filling is a dynamic, temporally regulated phase of seed development that determines the composition of storage reserves in mature seeds. Although the metabolic pathways responsible for storage reserve synthesis such as carbohydrates, oils, and proteins are known, little is known about their regulation. Protein phosphorylation is a ubiquitous form of regulation that influences many aspects of dynamic cellular behavior in plant biology. Here a systematic study has been conducted on five sequential stages (2, 3, 4, 5, and 6 weeks after flowering) of seed development in oilseed rape (Brassica napus L. Reston) to survey the presence and dynamics of phosphoproteins. High resolution two-dimensional gel electrophoresis in combination with a phosphoprotein-specific Pro-Q Diamond phosphoprotein fluorescence stain revealed approximately 300 phosphoprotein spots. Of these, quantitative expression profiles for 234 high quality spots were established, and hierarchical cluster analyses revealed the occurrence of six principal expression trends during seed filling. The identity of 103 spots was determined using LC-MS/MS. The identified spots represented 70 non-redundant phosphoproteins belonging to 10 major functional categories including energy, metabolism, protein destination, and signal transduction. Furthermore phosphorylation within 16 non-redundant phosphoproteins was verified by mapping the phosphorylation sites by LC-MS/MS. Although one of these sites was postulated previously, the remaining sites have not yet been reported in plants. Phosphoprotein data were assembled into a web database. Together this study provides evidence for the presence of a large number of functionally diverse phosphoproteins, including global regulatory factors like 14-3-3 proteins, within developing B. napus seed.
Highlights
Seed filling is a dynamic, temporally regulated phase of seed development that determines the composition of storage reserves in mature seeds
Extensive studies on seed development have firmly established that the components of storage reserve begin to accumulate and their relative levels are determined in the mature seed during a particular phase of seed development, referred to as seed filling [1, 4, 11, 12]
We have embarked on large scale phosphoproteomics study during seed filling in B. napus with the following objectives: (i) to obtain quantitative expression profiles of phosphoproteins through seed development, (ii) to generate a 2-DGE phosphoprotein reference map, (iii) to determine the phosphorylation sites of phosphoproteins, and (iv) to begin building resources for dissecting biological processes that might be regulated by reversible phosphorylation, including metabolism
Summary
2-DGE, two-dimensional gel electrophoresis; CBB, Coomassie Brilliant Blue; CV, coefficient of variation; FA, formic acid; PGK, phosphoglycerate kinase; Pro-Q DPS, Pro-Q Diamond phosphoprotein stain; WAF, weeks after flowering; TEMED, N,N,NЈ,NЈ-tetramethylethylenediamine; 2-D, two-dimensional; ACP, acyl-carrier-protein. Two major advantages of this stain are: 1) it can be used for global quantitative analysis of phosphoproteins as it binds directly to the phosphate moiety of phosphoproteins with high sensitivity and linearity regardless of phosphoamino acid and 2) the stain is fully compatible with other staining methods and modern MS Despite these recent advancements, phosphoproteins in plants have been rarely studied on a large scale basis (for a review, see Ref. 41). We have embarked on large scale phosphoproteomics study during seed filling in B. napus with the following objectives: (i) to obtain quantitative expression profiles of phosphoproteins through seed development, (ii) to generate a 2-DGE phosphoprotein reference map, (iii) to determine the phosphorylation sites of phosphoproteins, and (iv) to begin building resources for dissecting biological processes that might be regulated by reversible phosphorylation, including metabolism. This study extends the oilseed proteomics web-based database with a “Brassica phosphoproteomics” resource for the plant community
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