Table Of ContentMethods in
Molecular Biology 1789
Cláudia Pereira Editor
Plant Vacuolar
Traffi cking
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:
http://www.springer.com/series/7651
Plant Vacuolar Trafficking
Methods and Protocols
Edited by
Cláudia Pereira
Faculty of Sciences, University of Porto, Porto, Portugal
Editor
Cláudia Pereira
Faculty of Sciences
University of Porto
Porto, Portugal
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-7855-7 ISBN 978-1-4939-7856-4 (eBook)
https://doi.org/10.1007/978-1-4939-7856-4
Library of Congress Control Number: 2018942329
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Preface
Vacuoles occupy a large volume in a plant cell and have important storage, homeostatic, and
regulatory functions. Given the status of this organelle in the cell, it is crucial to understand
not only its biogenesis but also how proteins reach and accumulate in the plant vacuole.
Indeed, protein vacuolar trafficking is a major topic in the study of protein sorting in plants,
being the issue of several studies for several years. This volume of the Methods in Molecular
Biology series aims to provide the scientific community in this subject with a collection of a
significant number of state-of-the-art protocols, ranging from the design and preparation of
fusion proteins and expression vectors to a more detailed analysis involving different micros-
copy techniques. Plant Vacuolar Trafficking: Methods and Protocols will guide researchers
along the different chapters, including introductions to their respective topics, lists of the
necessary materials and reagents, and step-by-step and readily reproducible laboratory pro-
tocols. Cutting edge and comprehensive, Plant Vacuolar Trafficking: Methods and Protocols is
a highly valuable resource for researchers interested in learning more about this field.
Porto, Portugal Cláudia Pereira
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 GREEN FLUORESCENT SEED, to Evaluate Vacuolar
Trafficking in Arabidopsis Seeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Tomoo Shimada, Kentaro Fuji, Takuji Ichino, Ooi-Kock Teh, Yasuko Koumoto,
and Ikuko Hara-Nishimura
2 Detection of CRISPR/Cas9-Induced Genomic Fragment Deletions
in Barley and Generation of Homozygous Edited Lines via Embryogenic
Pollen Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Eszter Kapusi and Eva Stöger
3 Vacuolar Sorting Determinants: Isolation and Study . . . . . . . . . . . . . . . . . . . . . . 21
Bruno Peixoto, Susana Pereira, Cláudia Pereira, and José Pissarra
4 Vacuole-Targeted Proteins: Ins and Outs of Subcellular Localization Studies . . . 33
Inês Carqueijeiro, Liuda J. Sepúlveda, Angela Mosquera,
Richard Payne, Cyrielle Corbin, Nicolas Papon, Thomas Dugé de Bernonville,
Sébastien Besseau, Arnaud Lanoue, Gaëlle Glévarec, Marc Clastre,
Benoit St-Pierre, Lucia Atehortùa, Nathalie Giglioli-Guivarc’h,
Sarah E. O’Connor, Audrey Oudin, and Vincent Courdavault
5 Plant Cell Vacuoles: Staining and Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . 55
Giovanni Stefano, Luciana Renna, and Federica Brandizzi
6 Vacuolar Targeting and Characterization of Recombinant Antibodies . . . . . . . . . 65
Carolina Gabriela Ocampo and Silvana Petruccelli
7 Isolation of Vacuoles from the Leaves of the Medicinal
Plant Catharanthus roseus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Inês Carqueijeiro, Henrique Noronha, Sara Bettencourt, Joana G. Guedes,
Patrícia Duarte, Hernâni Gerós, and Mariana Sottomayor
8 Flow Cytometry and Fluorescence Microscopy as Tools for Structural
and Functional Analysis of Vacuoles Isolated from Yeast and Plant Cells . . . . . . . 101
Jorge M. P. Rodrigues, Cátia S. Pereira, Natacha Fontes, Hernâni Gerós,
and Manuela Côrte-Real
9 Live Cell Imaging and Confocal Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Luciana Renna, Giovanni Stefano, and Federica Brandizzi
10 Imaging Vacuolar Anthocyanins with Fluorescence Lifetime
Microscopy (FLIM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Alexandra Chanoca, Brian Burkel, Erich Grotewold, Kevin W. Eliceiri,
and Marisa S. Otegui
11 The Use of Drugs in the Study of Vacuole Morphology and Trafficking
to the Vacuole in Arabidopsis thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Ricardo Tejos, Claudio Osorio-Navarro, and Lorena Norambuena
vii
viii Contents
12 Use of Brefeldin A and Wortmannin to Dissect Post-Golgi Organelles
Related to Vacuolar Transport in Arabidopsis thaliana . . . . . . . . . . . . . . . . . . . . 155
Junpei Takagi and Tomohiro Uemura
13 Use Endosidin2 to Study Plant Exocytosis and Vacuolar Trafficking . . . . . . . . . . 167
Lei Huang and Chunhua Zhang
14 Visualization of RMRs (Receptor Membrane RING-H2) Dimerization
in Nicotiana benthamiana Leaves Using a Bimolecular Fluorescence
Complementation (BiFC) Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Alessandro Occhialini
15 Vacuole Dynamics in Rice Cells Invaded by the Blast Fungus
Magnaporthe oryzae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Kiersun Jones and Chang Hyun Khang
16 Characterization of Plant Glycoproteins: Analysis of Plant Glycopeptide
Mass Spectrometry Data with plantGlycoMS, a Package
in the R Statistical Computing Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Margaret R. Baker, Travers Ching, David L. Tabb, and Qing X. Li
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Contributors
Lucia atehortùa · Laboratorio de Biotecnología, Sede de Investigación Universitaria,
Universidad de Antioquia, Medellin, Colombia
Margaret r. Baker · Department of Molecular Biosciences and Bioengineering,
University of Hawaii at Manoa, Honolulu, HI, USA
thoMas Dugé De BernonviLLe · EA2106 “Biomolécules et Biotechnologies Végétales”,
UFR Sciences et Techniques, Université François-Rabelais de Tours, Tours, France
séBastien Besseau · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France
sara Bettencourt · CIBIO/InBIO—Centro de Investigação em Biodiversidade e
Recursos Genéticos, Universidade do Porto, Vairão, Portugal
FeDerica BranDizzi · MSU-DOE Plant Research Lab, Plant Biology Department,
Michigan State University, East Lansing, MI, USA; Department of Plant Biology,
Michigan State University, East Lansing, MI, USA
Brian BurkeL · Laboratory for Optical and Computational Instrumentation (LOCI),
University of Wisconsin-Madison, Madison, WI, USA
inês carqueijeiro · EA2106 Biomolécules et Biotechnologies Végétales, Département de
Biologie et Physiologie Végétales, UFR Pharmacologie, Université François-Rabelais de
Tours, Tours, France
aLexanDra chanoca · Department of Botany, University of Wisconsin-Madison, Madison,
WI, USA; Laboratory of Molecular and Cellular Biology, University of Wisconsin-
Madison, Madison, WI, USA
travers ching · Department of Molecular Biosciences and Bioengineering, University of
Hawaii at Manoa, Honolulu, HI, USA
Marc cLastre · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France
cyrieLLe corBin · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France
ManueLa côrte-reaL · Centre of Molecular and Environmental Biology, CBMA,
University of Minho, Braga, Portugal
vincent courDavauLt · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR
Sciences et Techniques, Université François-Rabelais de Tours, Tours, France
Patrícia Duarte · CIBIO/InBIO—Centro de Investigação em Biodiversidade e Recursos
Genéticos, Universidade do Porto, Vairão, Portugal
kevin W. eLiceiri · Laboratory of Molecular and Cellular Biology, University of
Wisconsin-Madison, Madison, WI, USA; Laboratory for Optical and Computational
Instrumentation (LOCI), University of Wisconsin-Madison, Madison, WI, USA
natacha Fontes · Sogrape Vinhos, S .A ., Research and Development Department,
Aldeia Nova, Avintes, Portugal
kentaro Fuji · Graduate School of Science, Kyoto University, Kyoto, Japan
ix
x Contributors
hernâni gerós · Centre of Molecular and Environmental Biology, CBMA, University of
Minho, Braga, Portugal; Centre of Biological Engineering, CEB, University of Minho,
Braga, Portugal; Centre for the Research and Technology of Agro-environmental and
Biological Sciences, CITAB—UMinho Pole, University of Minho, Braga, Portugal
nathaLie gigLioLi-guivarc’h · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR
Sciences et Techniques, Université François-Rabelais de Tours, Tours, France
gaëLLe gLévarec · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France
erich groteWoLD · Department of Biochemistry and Molecular Biology, Michigan State
University, East Lansing, MI, USA
joana g. gueDes · CIBIO/InBIO—Centro de Investigação em Biodiversidade e Recursos
Genéticos, Universidade do Porto, Vairão, Portugal; Departamento de Biologia,
Faculdade de Ciências da, Universidade do Porto, Porto, Portugal; Instituto de Ciências
Biomédicas Abel Salazar, Porto, Portugal
ikuko hara-nishiMura · Graduate School of Science, Kyoto University, Kyoto, Japan;
Faculty of Science and Engineering, Konan University, Kobe, Japan
Lei huang · Department of Botany and Pathology, Purdue University, West Lafayette, IN,
USA
takuji ichino · Graduate School of Science, Kyoto University, Kyoto, Japan
kiersun jones · Department of Plant Biology, University of Georgia, Athens, GA, USA
eszter kaPusi · Department of Applied Genetics and Cell Biology, University of Natural
Resources and Life Sciences, Vienna, Austria
chang hyun khang · Department of Plant Biology, University of Georgia, Athens, GA,
USA
yasuko kouMoto · Graduate School of Science, Kyoto University, Kyoto, Japan
arnauD Lanoue · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France
qing x. Li · Department of Molecular Biosciences and Bioengineering, University of
Hawaii at Manoa, Honolulu, HI, USA
angeLa Mosquera · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France; Laboratorio de
Biotecnología, Sede de Investigación Universitaria, Universidad de Antioquia, Medellin,
Colombia
Lorena noraMBuena · Department of Biology, Faculty of Sciences, Plant Molecular
Biology Centre, Universidad de Chile, Santiago, Chile
henrique noronha · Centro de Investigação e de Tecnologias Agro-Ambientais e
Biológicas, Vila Real, Portugal; Centro de Biologia Molecular e Ambiental (CBMA),
Departamento de Biologia, Universidade do Minho, Braga, Portugal
caroLina gaBrieLa ocaMPo · CIDCA-CCT-La Plata CONICET, Facultad de Ciencias
Exactas, Universidad Nacional de La Plata (UNLP), La Plata, Argentina
aLessanDro occhiaLini · Department of Food Science, University of Tennessee, Food Safety
and Processing Building, Knoxville, TN, USA
sarah e. o’connor · Department of Biological Chemistry, The John Innes Centre,
Norwich Research Park, Norwich, UK
cLauDio osorio-navarro · Department of Biology, Faculty of Sciences, Plant Molecular
Biology Centre, Universidad de Chile, Santiago, Chile
Contributors xi
Marisa s. otegui · Department of Botany, University of Wisconsin-Madison, Madison,
WI, USA; Laboratory of Molecular and Cellular Biology, University of
Wisconsin- Madison, Madison, WI, USA; Department of Genetics, University of
Wisconsin-Madison, Madison, WI, USA
auDrey ouDin · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France
nicoLas PaPon · EA3142 “Groupe d’Etude des Interactions Hôte-Pathogène”,
Université d’Angers, Angers, France
richarD Payne · Department of Biological Chemistry, The John Innes Centre, Norwich,
UK
Bruno Peixoto · Department of Biology, Faculty of Sciences, BioISI—Biosystems and
Integrative Sciences Institute, University of Porto, Porto, Portugal
cátia s. Pereira · Centre of Molecular and Environmental Biology, CBMA, University of
Minho, Braga, Portugal; Centre of Biological Engineering, CEB, University of Minho,
Braga, Portugal
cLáuDia Pereira · Faculty of Sciences, University of Porto, Porto, Portugal
susana Pereira · Department of Biology, Faculty of Sciences, BioISI—Biosystems and
Integrative Sciences Institute, University of Porto, Porto, Portugal
siLvana PetrucceLLi · CIDCA-CCT-La Plata CONICET, Facultad de Ciencias Exactas,
Universidad Nacional de La Plata (UNLP), La Plata, Argentina
josé Pissarra · Department of Biology, Faculty of Sciences, BioISI—Biosystems and
Integrative Sciences Institute, University of Porto, Porto, Portugal
Luciana renna · MSU-DOE Plant Research Lab, Plant Biology Department,
Michigan State University, East Lansing, MI, USA; Department of Plant Biology,
Michigan State University, East Lansing, MI, USA
jorge M. P. roDrigues · Centre of Molecular and Environmental Biology, CBMA,
University of Minho, Braga, Portugal
LiuDa j. sePúLveDa · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France; Laboratorio de
Biotecnología, Sede de Investigación Universitaria, Universidad de Antioquia, Medellin,
Colombia
toMoo shiMaDa · Graduate School of Science, Kyoto University, Kyoto, Japan
Mariana sottoMayor · CIBIO/InBIO—Centro de Investigação em Biodiversidade e
Recursos Genéticos, Universidade do Porto, Vairão, Portugal; Departamento de Biologia,
Faculdade de Ciências da, Universidade do Porto, Porto, Portugal
giovanni steFano · MSU-DOE Plant Research Lab, Michigan State University, East
Lansing, MI, USA; Department of Plant Biology, Michigan State University, East
Lansing, MI, USA
eva stöger · Department of Applied Genetics and Cell Biology, University of Natural
Resources and Life Sciences, Vienna, Austria
Benoit st-Pierre · EA2106 “Biomolécules et Biotechnologies Végétales”, UFR Sciences et
Techniques, Université François-Rabelais de Tours, Tours, France
DaviD L. taBB · Division of Molecular Biology and Human Genetics, Faculty of Medicine
and Health Sciences, SA MRC Centre for TB Research, DST/NRF Centre of Excellence
for Biomedical TB Research, Stellenbosch University, Cape Town, South Africa
