Abstract
To date, less than 150 proteins have been located to plant peroxisomes, indicating that unbiased large-scale approaches such as experimental proteome research are required to uncover the remaining yet unknown metabolic functions of this organelle as well as its regulatory mechanisms and membrane proteins. For experimental proteome research, Arabidopsis thaliana is the model plant of choice and an isolation methodology that obtains peroxisomes of sufficient yield and high purity is vital for research on this organelle. However, organelle enrichment is more difficult from Arabidopsis when compared to other plant species and especially challenging for peroxisomes. Leaf peroxisomes from Arabidopsis are very fragile in aqueous solution and show pronounced physical interactions with chloroplasts and mitochondria in vivo that persist in vitro and decrease peroxisome purity. Here, we provide a detailed protocol for the isolation of Arabidopsis leaf peroxisomes using two different types of density gradients (Percoll and sucrose) sequentially that yields approximately 120 μg of peroxisome proteins from 60 g of fresh leaf material. A method is also provided to assess the relative purity of the isolated peroxisomes by immunoblotting to allow selection of the purest peroxisome isolates. To enable the analysis of peroxisomal membrane proteins, an enrichment strategy using sodium carbonate treatment of isolated peroxisome membranes has been adapted to suit isolated leaf peroxisomes and is described here.
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
Similar content being viewed by others
Abbreviations
- CE:
-
Crude extract
- FW:
-
Fresh weight
- GB:
-
Grinding buffer
- HPR:
-
Hydroxypyruvate reductase
- IS:
-
Internal standard
- LP-P1/2:
-
First/second purified leaf peroxisome fraction
- RbcL:
-
RuBisCO large subunit
- SHMT:
-
Serine hydroxymethyltransferase
- TE:
-
Tricine–EDTA
References
Hu J, Baker A, Bartel B et al (2012) Plant peroxisomes: biogenesis and function. Plant Cell 24:2279–2303
Kaur N, Reumann S, Hu J (2009) Peroxisome Biogenesis and Function. In: Somerville CR, Meyerowitz EM, (eds) The Arabidopsis book. Rockville, MD: The American Society of Plant Biologists. pp 1–41
Kim HU, van Oostende C, Basset GJ et al (2008) The AAE14 gene encodes the Arabidopsis o-succinylbenzoyl-CoA ligase that is essential for phylloquinone synthesis and photosystem-I function. Plant J 54:272–283
Tanabe Y, Maruyama J, Yamaoka S et al (2011) Peroxisomes are involved in biotin biosynthesis in Aspergillus and Arabidopsis. J Biol Chem 286:30455–30461
Widhalm JR, Ducluzeau AL, Buller NE et al (2012) Phylloquinone (vitamin K(1)) biosynthesis in plants: two peroxisomal thioesterases of Lactobacillales origin hydrolyze 1,4-dihydroxy-2-naphthoyl-CoA. Plant J 71:205–215
Cooper TG, Beevers H (1969) Beta oxidation in glyoxysomes from castor bean endosperm. J Biol Chem 244:3514–3520
Harrison-Lowe N, Olsen LJ (2006) Isolation of glyoxysomes from pumpkin cotyledons. Curr Protoc Cell Biol 3(19):1–8
Lopez-Huertas E, Sandalio LM, Del Rio LA (1995) Integral membrane polypeptides of pea leaf peroxisomes: Characterization and response to plant stress. Plant Physiol Biochem 33:295–302
Yu C, Huang AH (1986) Conversion of serine to glycerate in intact spinach leaf peroxisomes: role of malate dehydrogenase. Arch Biochem Biophys 245:125–133
AGI (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815
Reumann S, Babujee L, Ma C et al (2007) Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms. Plant Cell 19:3170–3193
Reumann S, Quan S, Aung K et al (2009) In-depth proteome analysis of Arabidopsis leaf peroxisomes combined with in vivo subcellular targeting verification indicates novel metabolic and regulatory functions of peroxisomes. Plant Physiol 150:125–143
Fukao Y, Hayashi M, Nishimura M (2002) Proteomic analysis of leaf peroxisomal proteins in greening cotyledons of Arabidopsis thaliana. Plant Cell Physiol 43:689–696
Fukao Y, Hayashi M, Hara-Nishimura I et al (2003) Novel glyoxysomal protein kinase, GPK1, identified by proteomic analysis of glyoxysomes in etiolated cotyledons of Arabidopsis thaliana. Plant Cell Physiol 44:1002–1012
Quan S, Yang P, Cassin-Ross G et al (2013) Proteome analysis of peroxisomes from etiolated Arabidopsis seedlings identifies a peroxisomal protease involved in beta-oxidation and development. Plant Physiol 163:1518–1538
Eubel H, Meyer EH, Taylor NL et al (2008) Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes. Plant Physiol 148:1809–1829
Bussell JD, Behrens C, Ecke W et al (2013) Arabidopsis peroxisome proteomics. Front Plant Sci 4:101
Lingner T, Kataya AR, Antonicelli GE et al (2011) Identification of novel plant peroxisomal targeting signals by a combination of machine learning methods and in vivo subcellular targeting analyses. Plant Cell 23:1556–1572
Lingner T, Kataya AR, Reumann S (2012) Experimental and statistical post-validation of positive example EST sequences carrying peroxisome targeting signals type 1 (PTS1). Plant Signal Behav 7
Reumann S, Singhal R (2014) Isolation of leaf peroxisomes from Arabidopsis for organelle proteome analyses. Methods Mol Biol 1072:541–552
Fujiki Y, Hubbard AL, Fowler S et al (1982) Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol 93:97–102
Ma C, Haslbeck M, Babujee L et al (2006) Identification and characterization of a stress-inducible and a constitutive small heat-shock protein targeted to the matrix of plant peroxisomes. Plant Physiol 141:47–60
Hoagland DR, Arnon DI (1950) The waterculture method for growing plants without soil. California Agricultural Experiment Station Circular 347:1–32
Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute-solution in the presence of detergents and lipids. Anal Biochem 138:141–143
Reumann S (2011) Toward a definition of the complete proteome of plant peroxisomes: where experimental proteomics must be complemented by bioinformatics. Proteomics 11: 1764–1779
Timm S, Florian A, Jahnke K et al (2011) The hydroxypyruvate-reducing system in Arabidopsis: multiple enzymes for the same end. Plant Physiol 155:694–705
Timm S, Nunes-Nesi A, Parnik T et al (2008) A cytosolic pathway for the conversion of hydroxypyruvate to glycerate during photorespiration in Arabidopsis. Plant Cell 20:2848–2859
Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Acknowledgments
The research is supported by the Marie Curie Initial Training Networks (ITN) action PerFuMe (project number 316723).
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
Reumann, S., Lisik, P. (2017). Isolation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane. 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_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6533-5_8
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