title: Purification and proteomic characterization of plastids from Brassica napus developing embryos.
abstract: Plastids are functionally and structurally diverse organelles responsible for numerous biosynthetic reactions within the plant cell. Plastids from embryos have a range of properties depending upon the plant source but compared to other plastid types are poorly understood and therefore, we term them embryoplasts. Isolating intact plastids from developing embryos is challenging due to large starch granules within the stroma and the prevalence of nonplastid, storage organelles (oil bodies and protein storage vacuoles) which compromise plastid integrity and purity, respectively. To characterize rapeseed embryoplasts it was necessary to develop an improved isolation procedure. A new method is presented for the isolation of intact plastids from developing embryos of Brassica napus seeds. Intactness and purity of embryoplast preparations was determined using phase-contrast and transmission electron microscopy, immunoblotting, and multidimensional protein identification technology (MudPIT) MS/MS. Eighty nonredundant proteins were identified by MudPIT analysis of embryoplast preparations. Approximately 53% of these proteins were components of photosystem, light harvesting, cytochrome b/f, and ATP synthase complexes, suggesting ATP and NADPH production are important functions for this plastid type.
authors.affiliation: Department of Biochemistry, Life Science Center, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Renuka
authors.initials: R
authors.lastname: Jain
authors.forename: Vesna
authors.initials: V
authors.lastname: Katavic
authors.forename: Ganesh Kumar
authors.initials: GK
authors.lastname: Agrawal
authors.forename: Victor M
authors.initials: VM
authors.lastname: Guzov
authors.forename: Jay J
authors.initials: JJ
authors.lastname: Thelen
mesh_terms: D029688:Brassica napus / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D046529:Microscopy, Electron, Transmission; D045322:Photosynthetic Reaction Center Complex Proteins / Q000032:analysis / Q000302:isolation & purification; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D018087:Plastids / Q000378:metabolism* / Q000648:ultrastructure; D040901:Proteomics / Q000379:methods*; D012639:Seeds / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins