Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Natesan SK, Sullivan JA, Gray JC (2005) Stromules: a characteristic cell-specific feature of plastid morphology. J Exp Bot 56:787–797
Boyle SA, Hemmingsen SM, Dennis DT (1986) Uptake and processing of the precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase by leucoplasts from the endosperm of developing castor oil seeds. Plant Physiol 81:817–822
Boyle SA, Hemmingsen SM, Dennis DT (1990) Energy requirement for the import of protein into plastids from developing endosperm of Ricinus-communis L. Plant Physiol 92:151–154
Dennis DT (1989) Fatty acid biosynthesis in plastids. In: Boyer, DC, Shannon JC, Hardison RC, (eds) Physiology, biochemistry, and genetics of nongreen plastids. American Society of Plant Physiologists pp 120–129
Eastmond PJ, Dennis DT, Rawsthorne S (1997) Evidence that a malate inorganic phosphate exchange translocator imports carbon across the leucoplast envelope for fatty acid synthesis in developing castor seed endosperm. Plant Physiol 114:851–856
Miernyk JA (1989) Leucoplast isolation. In: Boyer DC, Shannon JC, Hardison RC, (eds) Physiology, biochemistry, and genetics of nongreen plastids. American Society of Plant Physiologists. pp 15–19
Negm FB, Cornel FA, Plaxton WC (1995) Suborganellar localization and molecular characterization of nonproteolytic degraded leukoplast pyruvate kinase from developing castor oil seeds. Plant Physiol 109:1461–1469
Shearer HL, Turpin DH, Dennis DT (2004) Characterization of NADP-dependent malic enzyme from developing castor oil seed endosperm. Arch Biochem Biophys 429:134–144
Smith RG, Gauthier DA, Dennis DT et al (1992) Malate- and pyruvate-dependent fatty acid synthesis in leucoplasts from developing castor endosperm. Plant Physiol 98:1233–1238
Wan JX, Blakeley SD, Dennis DT et al (1996) Transit peptides play a major role in the preferential import of proteins into leucoplasts and chloroplasts. J Biol Chem 271:31227–31233
Lara-Nunez A, Rodriguez-Sotres R (2004) Characterization of a dicarboxylate exchange system able to exchange pyrophosphate for L-malate in non-photosynthetic plastids from developing maize embryos. Plant Sci 166:1335–1343
Gupta R, Singh R (1996) Fatty acid synthesis in leucoplasts isolated from developing seeds of Brassica campestris. J Plant Biochem Biotechnol 5:127–130
Nagegowda DA, Gutensohn M, Wilkerson CG et al (2008) Two nearly identical terpene synthases catalyze the formation of nerolidol and linalool in snapdragon flowers. Plant J 55:224–239
Greenwood JS, Bewley JD (1982) Seed development in Ricinus-communis (castor bean).1. Descriptive morphology. Can J Bot 60:1751–1760
Eubel H, Heazlewood JL, Millar AH (2007) Isolation and subfractionation of plant mitochondria for proteomic analysis. Methods Mol Biol 355:49–62
Alban C, Joyard J, Douce R (1988) Preparation and characterization of envelope membranes from nongreen plastids. Plant Physiol 88:709–717
Echeverria E, Boyer C, Liu KC et al (1985) Isolation of amyloplasts from developing maize endosperm. Plant Physiol 77:513–519
Jain R, Katavic V, Agrawal GK et al (2008) Purification and proteomic characterization of plastids from Brassica napus developing embryos. Proteomics 8:3397–3405
Bollag DM, Edelstein SJ (1991) Protein concentration determination. In: Protein methods. Wiley-Liss, New York. pp 50–55
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Plaxton, W.C. (2017). Leucoplast Isolation and Subfractionation. In: Taylor, N., Millar, A. (eds) Isolation of Plant Organelles and Structures. Methods in Molecular Biology, vol 1511. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6533-5_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6533-5_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6531-1
Online ISBN: 978-1-4939-6533-5
eBook Packages: Springer Protocols