Chloroplast Research in Arabidopsis Methods and Protocols, Volume II
Chloroplasts are essential for the survival and flourishing of life on Earth. Over the years, chloroplast biology has been studied in a variety of different organisms, leading to the significant disadvantage that findings which were made by using differen
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. Introduction Plastoglobules are lipoprotein particles present in different plastid types such as proplastids, chloroplasts, chromoplasts, and gerontoplasts. Plastoglobules are mainly composed of isoprenoids, neutral lipids, and proteins (1). They are physically attached to thylakoids via the outer half of the lipid bilayer of the thylakoid, which surrounds plastoglobules (2). Long viewed as passive lipid droplets, plastoglobules have been receiving increasing attention in the last half decade. Indeed, proteomic studies of plastoglobules (3, 4) have demonstrated their active role in plastid biology. This idea is reinforced by the fact that plastoglobule size and number vary depending on plastid type and environmental conditions. Currently, a growing body of evidence suggests that plastoglobules are *Celine Besagni and Lucia Eugeni Piller have contributed equally to this chapter. R. Paul Jarvis (ed.), Chloroplast Research in Arabidopsis: Methods and Protocols, Volume II, Methods in Molecular Biology, vol. 775, DOI 10.1007/978-1-61779-237-3_12, © Springer Science+Business Media, LLC 2011
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involved in various metabolic pathways such as those leading to vitamin K (5), tocopherol (3, 6), and carotenoids (4), and in plant response to stress by accumulating antioxidants (e.g., tocopherols) and sequestrating toxic molecules (e.g., fatty acid phytyl esters) (7). In addition, studies of the plastid ultrastructure suggested that plastoglobule size and number are regulated in correlation with the fitness of the thylakoid membranes, putatively playing the role of a reservoir for thylakoid membrane lipids (1, 8). Besides their physiological roles, plastoglobules may present a biotechnological interest for molecular farming by providing a hydrophobic environment necessary for the production of certain proteins, associated with a simple purification procedure (3). The identification of components (proteins or lipids) specifically localized in plastoglobules relies on preparation of a plastoglobule fraction exempt of any contamination. Such contamination could originate either from light particles present in other compartments of the cell (e.g., oil bodies) or from plastid membranes. Thus, the preparation of pure plastoglobules depends on two critical steps: (1) isolation of intact plastids on Percoll gradients and (2) accurate separation of plastid membranes by flotation on sucrose gradients. The range of sucrose concentrations (5–45%) that is used for the second step allows the separation of plastoglobules from envelope membranes, which is generally not achieved with standard protocols designed for plastid membrane preparation. In addition, because plastoglobules are physically attached to thylakoids, the procedure must include a step to achieve a good separation of plastoglobules from thylakoids. Finally, the purity of the plastoglobule fraction is verified by immunoblotting, and plastoglobules are then available for proteomic analysis or other studies.
2. Materials 2.1. Arabidopsis Culture
1. U
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