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.

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

“…Most of the data available were related to chloroplasts (Armbruster et al, 2011;van Wijk and Baginsky, 2011), although some information was also available for nonphotosynthetic plastid structures such as amyloplasts (Andon et al, 2002;Balmer et al, 2006), etioplasts (von Zychlinski et al, 2005, proplastids (Baginsky et al, 2004), and embryoplasts (Demartini et al, 2011). The present inventory also provides information that complements previous articles published on the chromoplast proteome (bell pepper: Siddique et al, 2006;tomato: Barsan et al, 2010;and sweet orange: Zeng et al, 2011).…”

“…The spectral-count-based label free has been reported as allowing quantification of protein abundance with similar efficiency to isotope labeling (Zhu et al, 2009). This methodology had been applied in a number of other studies related to the plastid proteome (Bräutigam et al, 2008;Ferro et al, 2010;Demartini et al, 2011).…”

Integration of a constraint-based metabolic model of Brassica napus developing seeds with 13C-metabolic flux analysis

“…“2” was given at the level of physiological evidence, for example, a spontaneous non-enzymatic reaction and for B. napus chloroplast proteome (Demartini et al, 2011 ). “3” was used for biochemical evidence in Brassicaceae species other than B. napus using BRENDA (Schomburg et al, 2013 ), or for developing B. napus embryo transcriptome (Troncoso-Ponce et al, 2011 ) and B. napus embryo plastid proteome data (Demartini et al, 2011 ). “4” was the highest score awarded, and used for biochemical evidence (e.g., enzyme activity assayed) in B. napus according to the literature or BRENDA.…”

Plant proteomics in India and Nepal: current status and challenges ahead

“…Spectral counting based quantitative proteomic analysis of plastids from developing embryos and leaves of Brassica napus showed that plastids from leaves were rich in light reaction proteins while Calvin cycle enzymes were more prominent in plastids isolated from developing embryo (Demartini et al 2011). Interestingly, enzymes related to de novo fatty acid and amino acid biosynthesis were detected in embryoplasts but not in chloroplasts.…”