Isolation of Plastoglobules for Lipid Analyses

Plastoglobules are plastid compartments designed for the storage of neutral lipids. They share physical and structural characteristics with cytosolic lipid droplets. Hence, special care must be taken to avoid contamination by cytosolic lipid droplets during plastoglobule purification. We describe the isolation of pure plastoglobules from Arabidopsis thaliana leaves, and the methods we use to determine their lipid composition. After preparation of a crude chloroplast fraction, plastoglobules are isolated from plastid membranes by two steps of ultracentrifugation on discontinuous sucrose gradients. For lipid analyses, total lipids are then extracted by a standard chloroform-methanol protocol, and polar lipids are separated from neutral lipids by liquid-liquid extraction. While polar lipid classes are subsequently separated by thin-layer chromatography (TLC) with the classical Vitiello solvent mix, a double TLC development has to be performed for neutral lipids, to separate phytyl and steryl esters. Lipids are quantified by gas chromatography after conversion of the fatty acids into methyl esters.