Proteomic Comparison of Plastids from Developing Embryos and Leaves of Brassica napus

Abstract

Plastids are highly specialized organelles, responsible for photosynthesis and biosynthesis of various phytochemicals. To better understand plastid diversity and metabolism, a quantitative proteomic study of two plastid forms from Brassica napus (oilseed rape) was performed. Plastids were isolated from leaves (chloroplasts) of two-week-old plants and developing embryos (embryoplasts) three-weeks after flowering, using an approach avoiding protein storage vacuole contamination. Proteins from five different plastid preparations were prefractionated by SDS-PAGE and sectioned into multiple bands, and in-gel proteins were subjected to trypsin digestion. Tryptic peptides from each band were eluted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and spectra were searched against a comprehensive plant database. Proteins were quantified based on MS/MS spectral counting of unique, nonhomologous peptides. Functional classification and quantitative comparison of over 2000 redundant proteins (compiled to 675 nonredundant proteins) determined that light reaction proteins are more prominent in chloroplasts, while many Calvin cycle enzymes are more prominent in embryoplasts. Embryoplasts also contain a diversity of other metabolic enzymes undetected in chloroplasts. Many enzymes involved in de novo fatty acid and amino acid biosynthesis were detected in embryoplasts but not chloroplasts. Additionally, protein synthesis-related proteins were prominent in embryoplasts. Collectively, these results indicate that these two plastid types are distinct.