Table Of ContentMethods in
Molecular Biology 1511
Nicolas L. Taylor
A. Harvey Millar Editors
Isolation of
Plant Organelles
and Structures
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB , UK
For further volumes:
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Isolation of Plant Organelles
and Structures
Methods and Protocols
Edited by
Nicolas L. Taylor
The ARC Centre of Excellence in Plant Energy Biology and School of Chemistry and Biochemistry,
The University of Western Australia, Perth, Western Australia, Australia
A. Harvey Millar
The ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia,
Perth, Western Australia, Australia
Editors
Nicolas L. Taylor A. Harvey Millar
The ARC Centre of Excellence in Plant The ARC Centre of Excellence in Plant
Energy Biology and School of Chemistry Energy Biology
and Biochemistry The University of Western Australia
The University of Western Australia Perth, Western Australia
Perth, Western Australia Australia
Australia
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6531-1 ISBN 978-1-4939-6533-5 (eBook)
DOI 10.1007/978-1-4939-6533-5
Library of Congress Control Number: 2016955174
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Prefa ce
The isolation of plant organelles and subcellular structures has been the aim of plant scien-
tists for some time. Sam Granick’s pioneering work in the late 1930s isolated the fi rst sub-
cellular plant bodies and aimed to “isolate chloroplasts in as normal a state and with as little
contamination as possible” [1], an aim that many also aspire to today when isolating a
range of organelles or subcellular structures of interest. In addition, he “hoped that the
quantitative isolation of chloroplasts will open up a new and more fruitful fi eld of investiga-
tion, not only on the composition, structure and metabolism of these bodies, but also the
mechanism of photosynthesis” [1]. The methods presented here were developed and
refi ned with similar goals in mind, in that by being able to isolate subcellular structures the
research and understanding of various facets of compartmentalized function in plant cells
would be advanced. This book aims to bring together the major techniques used in the
isolation or enrichment of individual populations of organelles and other subcellular struc-
tures from plants. An expert in the isolation of each particular cellular component has
composed each chapter, and they have provided a step-by-step procedure aimed at research-
ers from all fi elds of plant science. It should suit those who regularly isolate subcellular
components as well as those whose research has led them to focus on a subcellular compart-
ment or a particular process for the fi rst time, thus producing the need to be able to isolate
it or enrich it for study.
Perth, WA, Australia Nicolas L. Taylor
A. Harvey Millar
Reference
1. Granick S (1938) Isolation of chloroplasts from higher plants. Am J Bot 25:558–561
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 T he Isolation of Plant Organelles and Structures in the Post-genomic Era. . . . 1
A. Harvey Millar and Nicolas L. Taylor
2 A pproaches to Characterize Organelle, Compartment, or Structure Purity. . . . 13
Stefanie J. Mueller , S ebastian N.W. Hoernstein , and Ralf Reski
PART I ISOLATION OF ORGANELLES AND ORGANELLE COMPARTMENTS
3 I solation of Nuclei and Nucleoli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1
Alison F. Pendle and Peter J. Shaw
4 I solation and Suborganellar Fractionation of Arabidopsis Chloroplasts. . . . . . . 4 5
Úrsula Flores-Pérez and Paul Jarvis
5 I solation of Chromoplasts and Suborganellar Compartments
from Tomato and Bell Pepper Fruit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Cristina Barsan , M arcel Kuntz , and Jean-Claude Pech
6 L eucoplast Isolation and Subfractionation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
William C. Plaxton
7 I solation of Mitochondria, Their Sub-Organellar Compartments,
and Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 3
Owen Duncan , A . Harvey Millar , and Nicolas L. Taylor
8 I solation of Arabidopsis Leaf Peroxisomes and the Peroxisomal Membrane . . . 97
Sigrun Reumann and Piotr Lisik
9 Isolation of Vacuoles and the Tonoplast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Jan Zouhar
10 I solation of Endoplasmic Reticulum and Its Membrane. . . . . . . . . . . . . . . . . . 1 19
G. Eric Schaller
11 E nrichment of Golgi Membranes from T riticum aestivum
(Wheat) Seedlings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Wei Zeng , B erit Ebert , H arriet T. Parsons , C arsten Rautengarten ,
Antony Bacic , and Joshua L. Heazlewood
12 I solation of Autolysosomes from Tobacco BY-2 Cells . . . . . . . . . . . . . . . . . . . 1 51
Chihiro Takatsuka , Yuko Inoue-Aono , and Yuji Moriyasu
13 I solation of Protein Storage Vacuoles and Their Membranes . . . . . . . . . . . . . . 1 63
Tomoo Shimada and Ikuko Hara-Nishimura
vii
viii Contents
PART II ISOLATION OF OTHER STRUCTURES AND COMPARTMENTS
14 I solation of the Cell Wall. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Hervé Canut , C écile Albenne , and Elisabeth Jamet
15 I solation of Plasmodesmata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Christine Faulkner and Emmanuelle M.F. Bayer
16 Isolation of Plasma Membrane and Plasma Membrane Microdomains . . . . . . . 199
Anzu Minami, Daisuke Takahashi, Yukio Kawamura,
and Matsuo Uemura
17 E nrichment of the Plant Cytosolic Fraction. . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Jeemeng Lao , A ndreia M. Smith-Moritz , J ennifer C. Mortimer ,
and Joshua L. Heazlewood
18 I solation of Apoplast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Erik Andreasson , K ibrom B. Abreha , and Svante Resjö
19 I solation of Cytosolic Ribosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 41
Hanna Klang Å rstrand and Maria V. Turkina
20 I solation of Plastid Ribosomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Kenichi Yamaguchi
21 I solation of Mitochondrial Ribosomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Adam J. Carroll
22 I solation of Microtubules and Microtubule-Associated Proteins. . . . . . . . . . . . 2 81
Takahiro Hamada and Seiji Sonobe
23 I solation of Actin and Actin-Binding Proteins. . . . . . . . . . . . . . . . . . . . . . . . . 291
Etsuo Yokota
24 P urification of 26S Proteasomes and Their Subcomplexes from Plants. . . . . . . 3 01
Richard S. Marshall , D avid C. Gemperline , and Richard D. Vierstra
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
Contributors
KIBROM B. ABREHA • Department of Plant Protection Biology, S wedish University
of Agricultural Sciences , A lnarp, S weden
CÉCILE ALBENNE • Laboratoire de Recherche en Sciences Végétales, U niversité de Toulouse,
CNRS, UPS , T olosan , F rance
ERIK ANDREASSON • Department of Plant Protection Biology, S wedish University
of Agricultural Sciences , A lnarp, S weden
HANNA KLANG ÅRSTRAND • Division of Cell Biology, Department of Clinical
and Experimental Medicine, L inköping University , L inköping, S weden
CRISTINA BARSAN • Génomique et Biotechnologie des Fruits, INRA,INP-ENSA Toulouse,
Université de Toulouse , C astanet-Tolosan, F rance
ANTONY BACIC • The ARC Centre of Excellence in Plant Cell Walls, School of Biosciences,
The University of Melbourne , M elbourne, V IC , A ustralia
EMMANUELLE M.F. BAYER • Laboratory of Membrane Biogenesis, U niversité Bordeaux ,
Villenave d’Ornon, F rance
HERVÉ CANUT • Laboratoire de Recherche en Sciences Végétales, U niversité de Toulouse,
CNRS, UPS , T olosan , F rance
ADAM J. CARROLL • The ARC Centre of Excellence for Translational Photosynthesis,
The Australian National University , C anberra, A CT, A ustralia
OWEN DUNCAN • The ARC Centre of Excellence in Plant Energy Biology,
The University of Western Australia , P erth , W A, A ustralia
BERIT EBERT • The ARC Centre of Excellence in Plant Cell Walls, School of Biosciences,
The University of Melbourne , M elbourne, V IC , A ustralia; Department of Biochemistry,
University of Cambridge, Cambridge, UK
CHRISTINE FAULKNER • John Innes Centre, Norwich Research Park , N orwich, U K
ÚRSULA FLORES-PÉREZ • Department of Plant Sciences, U niversity of Oxford , O xford , U K
DAVID C. GEMPERLINE • Department of Genetics, University of Wisconsin – Madison , WI , USA
TAKAHIRO HAMADA • Department of Life Sciences, Graduate School of Arts and Sciences,
The University of Tokyo , T okyo, J apan
IKUKO HARA-NISHIMURA • Department of Botany, Graduate School of Science,
Kyoto University , K yoto, J apan
JOSHUA L. HEAZLEWOOD • Joint BioEnergy Institute and Biological Systems and
Engineering Division, L awrence Berkeley National Laboratory , B erkeley , C A , U SA;
School of BioSciences, The University of Melbourne, Melbourne, VIC, Australia
SEBASTIAN N. W. HOERNSTEIN • Plant Biotechnology, Faculty of Biology,
University of Freiburg , F reiburg, G ermany
YUKO INOUE-AONO • Graduate School of Science and Engineering, S aitama University ,
Saitama, J apan
ELISABETH JAMET • Laboratoire de Recherche en Sciences Végétales, U niversité de Toulouse,
CNRS, UPS , T olosan , F rance
PAUL JARVIS • Department of Plant Sciences, U niversity of Oxford , O xford , U K
YUKIO KAWAMURA • United Graduate School of Agricultural Sciences and Cryobiofrontier
Research Center , I wate University , M orioka , J apan
ix
x Contributors
MARCEL KUNTZ • Laboratoire de Physiologie Cellulaire Végétale, C EA/CNRS/INRA/
Université Grenoble-Alpes , G renoble, F rance
JEEMENG LAO • Joint BioEnergy Institute and Biological Systems and Engineering Division,
Lawrence Berkeley National Laboratory , B erkeley , C A , U SA
PIOTR LISIK • Biocentre Klein Flottbek, University of Hamburg, H amburg, G ermany
RICHARD S . M ARSHALL • Department of Genetics, U niversity of Wisconsin– M adison , W I , U SA;
Department of Biology, Washington University in St Louis, MO, USA
A. HARVEY MILLAR • The ARC Centre of Excellence in Plant Energy Biology, The University
of Western Australia , P erth , W A, A ustralia
ANZU MINAMI • Bioscience and Biotechnology Center, N agoya University , N agoya , J apan
YUJI MORIYASU • Graduate School of Science and Engineering, S aitama University ,
Saitama, J apan
JENNIFER C. MORTIMER • Joint BioEnergy Institute and Biological Systems and Engineering
Division, Lawrence Berkeley National Laboratory , B erkeley , C A , U SA
STEFANIE J. MUELLER • Plant Biotechnology, Faculty of Biology, U niversity of Freiburg ,
Freiburg, G ermany
HARRIET T. PARSONS • Joint BioEnergy Institute and Biological Systems and Engineering
Division, L awrence Berkeley National Laboratory , B erkeley , C A , USA
JEAN-CLAUDE PECH • Génomique et Biotechnologie des Fruits, INRA, INP-ENSA Toulouse,
Université de Toulouse , T olosan , F rance
ALISON F. PENDLE • Department of Cell and Developmental Biology, John Innes Centre,
Norwich Research Park , N orwich, U K
WILLIAM C. PLAXTON • Department of Biology, Q ueen’s University , K ingston, O N , C anada
CARSTEN RAUTENGARTEN • The ARC Centre of Excellence in Plant Cell Walls,
School of Biosciences, T he University of Melbourne , M elbourne, V IC , A ustralia;
Department of Biochemistry, University of Cambridge, Cambridge, UK
SVANTE RESJÖ • Department of Plant Protection Biology, S wedish University of Agricultural
Sciences , A lnarp, S weden
RALF RESKI • Plant Biotechnology, Faculty of Biology, U niversity of Freiburg , F reiburg,
Germany ; B IOSS Centre for Biological Signalling Studies, U niversity of Freiburg ,
Freiburg, G ermany ; F RIAS Freiburg Institute for Advanced Studies, U niversity
of Freiburg , F reiburg, G ermany; TIP Trinational Institute for Plant Research,
University of Freiburg, Freiburg, Germany
SIGRUN REUMANN • Biocentre Klein Flottbek, U niversity of Hamburg , H amburg, G ermany ;
Centre for Organelle Research, U niversity of Stavanger , S tavanger, N orway
G. E RIC S CHALLER • Department of Biological Sciences, Dartmouth College , Hanover , NH , USA
PETER J. SHAW • Department of Cell and Developmental Biology, John Innes Centre,
Norwich Research Park , N orwich, U K
TOMOO SHIMADA • Department of Botany, Graduate School of Science, K yoto University ,
Kyoto, J apan
ANDREIA M. SMITH-MORITZ • Joint BioEnergy Institute and Biological Systems and
Engineering Division, Lawrence Berkeley National Laboratory , B erkeley , C A , U SA
SEIJI SONOBE • Department of Life Sciences, Graduate School of Life Sciences,
University of Hyogo , H yogo, J apan
DAISUKE TAKAHASHI • United Graduate School of Agricultural Sciences and Cryobiofrontier
Research Center , I wate University , M orioka , J apan
CHIHIRO TAKATSUKA • Tokai University Junior College , S hizuoka, J apan
Contributors xi
NICOLAS L. TAYLOR • The ARC Centre of Excellence in Plant Energy Biology and School
of Chemistry and Biochemistry , T he University of Western Australia , P erth , W A,
Australia
MARIA V. TURKINA • Division of Cell Biology, Department of Clinical and Experimental
Medicine, L inköping University , L inköping, S weden
MATSUO UEMURA • United Graduate School of Agricultural Sciences and Cryobiofrontier
Research Center , I wate University , M orioka , J apan
RICHARD D. VIERSTRA • Department of Genetics, University of Wisconsin – Madison, WI , USA
KENICHI YAMAGUCHI • Division of Biochemistry, Faculty of Fisheries, N agasaki University ,
Nagasaki, J apan
ETSUO YOKOTA • Department of Life Sciences, Graduate School of Life Sciences, U niversity
of Hyogo , H yogo, J apan
WEI ZENG • The ARC Centre of Excellence in Plant Cell Walls, School of Biosciences,
The University of Melbourne , M elbourne, V IC , A ustralia
JAN ZOUHAR • Centro de Biotecnología y Genómica de Plantas , U niversidad Politécnica de
Madrid , P ozuelo de Alarcón, S pain
