Leucoplast Isolation and Subfractionation.

Leucoplasts are colorless plastids of nonphotosynthetic plant tissues that support a variety of anabolic roles, particularly the biosynthesis of long-chain fatty acids in storage tissues of developing oil seeds. They also perform other important metabolic functions including the biosynthesis of amino acids and tetrapyrrole compounds. Leucoplasts use a complex set of membrane carriers and channels to actively translocate nuclear-encoded precursor proteins from the cytosol, while exchanging various metabolites with the cytosol. Leucoplast purification is a necessary prerequisite for detailed studies of their soluble (stromal) and membrane (envelope) (phospho)proteomes, as well as for achieving a detailed understanding of their metabolic capabilities, transport processes, and biogenesis. This chapter describes protocols for leucoplast purification from endosperm of developing castor oil seeds, and their subsequent subfractionation into envelope membrane and soluble stromal compartments for biochemical analysis.