Table Of ContentMethods in
Molecular Biology 1043
Mark P. Running Editor
G Protein-Coupled
Receptor Signaling
in Plants
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
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G Protein-Coupled Receptor
Signaling in Plants
Methods and Protocols
Edited by
Mark P. Running
Department of Biology, University of Louisville, Louisville, KY, USA
Editor
Mark P. Running
Department of Biology
University of Louisville
Louisville, KY , U SA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-531-6 ISBN 978-1-62703-532-3 (eBook)
DOI 10.1007/978-1-62703-532-3
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Preface
Plants, like other multicellular organisms, rely on signal transduction for cell–cell commu-
nication and responses to the environment. One prominent mechanism for signaling is the
use of small GTPases as on/off switches. In plants, heterotrimeric G proteins, consisting of
an alpha, beta, and gamma subunits, and Rop GTPases, which are Rho-related proteins in
plants, play a role in a myriad of developmental, hormonal, and environmental responses.
In addition, Rab-GTPases regulate intracellular traffi cking. This book describes methods
used in the study of small GTPases and related proteins.
In eukaryotes, heterotrimeric G proteins are associated with G protein-coupled recep-
tors (GPCRs). Remarkably, while GPCRs are highly prevalent in animals and yeast, very
few candidate GPCRs have been identifi ed in plants. One major issue is that GPCRs are not
well conserved in sequence. The chapter by Gookin and Bendtsen (Chapter 1 ) seeks to
address this by using bioinformatics approaches to identify proteins matching GPCR char-
acteristics. There is also a comparative dearth in the number of heterotrimeric G protein
subunits present in plant genomes. Despite this, heterotrimeric G proteins play roles in
several key plant processes, including several described in this book. In contrast, there are
several dozen Rab-GTPases present in plants, the function of each of which is just begin-
ning to be understood.
Finally, most G proteins are subject to lipid modifi cation, which serves to facilitate
membrane association and to promote protein–protein interactions. Heterotrimeric G pro-
tein alpha and gamma subunits, Rop-GTPases, and Rab-GTPases are all subject to the
addition to one or more lipid moieties. Several chapters cover how to identify these lipid
modifi cations, which include myristoylation, acylation, and prenylation. Further studies
into the roles of small GTPases will help elucidate several key processes in plants.
Louisville, KY , USA Mark P. Running
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Topology Assessment, G Protein-Coupled Receptor (GPCR) Prediction,
and In Vivo Interaction Assays to Identify Plant Candidate GPCRs. . . . . . . . . . 1
Timothy E. Gookin and Jannick D. Bendtsen
2 Measurement of GTP-Binding and GTPase Activity
of Heterotrimeric Gα Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Swarup Roy Choudhury, Corey S. Westfall, Dieter Hackenberg,
and Sona Pandey
3 Biochemical Analysis of the Interaction Between Phospholipase Dα1
and GTP-Binding Protein α-Subunit from Arabidopsis thaliana . . . . . . . . . . . . 21
Jian Zhao and Xuemin Wang
4 Analysis of Cell Division and Cell Elongation in the Hypocotyls
of Arabidopsis Heterotrimeric G Protein Mutants. . . . . . . . . . . . . . . . . . . . . . . 37
Zhaoqing Jin, Wellington Muchero, and Jin-Gui Chen
5 Aequorin Luminescence-Based Functional Calcium Assay
for Heterotrimeric G-Proteins in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Kiwamu Tanaka, Jeongmin Choi, and Gary Stacey
6 Methods for Analysis of Disease Resistance and the Defense Response
in Arabidopsis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Guojing Li, Xiujuan Zhang, Dongli Wan, Shuqun Zhang, and Yiji Xia
7 Fusarium oxysporum Infection Assays in Arabidopsis . . . . . . . . . . . . . . . . . . . . . 67
Yuri Trusov, David Chakravorty, and Jose Ramon Botella
8 Analysis of Unfolded Protein Response in Arabidopsis. . . . . . . . . . . . . . . . . . . . 73
Yani Chen and Federica Brandizzi
9 Functional Analysis of Heterotrimeric G Proteins in Chloroplast
Development in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Wenjuan Wu and Jirong Huang
10 G Protein Signaling in UV Protection: Methods
for Understanding the Signals in Young Etiolated Seedlings . . . . . . . . . . . . . . . 89
Danielle A. Orozco-Nunnelly, Lon S. Kaufman, and Katherine M. Warpeha
11 Functional Analysis of Small Rab GTPases in Cytokinesis
in Arabidopsis thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Xingyun Qi and Huanquan Zheng
12 In Vivo Localization in Arabidopsis Protoplasts and Root Tissue. . . . . . . . . . . . 113
Myoung Hui Lee, Yongjik Lee, and Inhwan Hwang
vii
viii Contents
13 Analysis of Protein Prenylation and S-Acylation Using
Gas Chromatography–Coupled Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . 121
Nadav Sorek, Amir Akerman, and Shaul Yalovsky
14 In Vitro Myristoylation Assay of Arabidopsis Proteins . . . . . . . . . . . . . . . . . . . . 135
Xuehui Feng, Wan Shi, Xuejun Wang, and Mark P. Running
15 Assaying Protein S-Acylation in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Piers A. Hemsley
16 In Vitro Prenylation Assay of Arabidopsis Proteins . . . . . . . . . . . . . . . . . . . . . . 147
Wan Shi, Qin Zeng, and Mark P. Running
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Contributors
AMIR AKERMAN • Department of Molecular Biology and Ecology of Plants, George S. Wise
Faculty of Life Sciences, Tel Aviv University , Tel Aviv , Israel
JANNICK D. BENDTSEN • CLC bio , Aarhus N , Denmark
JOSE RAMON BOTELLA • Plant Genetic Engineering Laboratory , School of Agriculture
and Food Sciences, University of Queensland , Brisbane, QLD , Australia
FEDERICA BRANDIZZI • Department of Energy Plant Research Laboratory , Michigan State
University , East Lansing, MI , USA ; Department of Plant Biology , Michigan State
University , East Lansing, MI , USA
DAVID CHAKRAVORTY • Plant Genetic Engineering Laboratory , School of Agriculture
and Food Sciences, University of Queensland , Brisbane, QLD , Australia
JIN-GUI CHEN • Biosciences Division, Oak Ridge National Laboratory , Oak Ridge, TN , USA
YANI CHEN • Department of Energy Plant Research Laboratory , Michigan State University ,
East Lansing, MI , USA ; Department of Plant Biology , Michigan State University , East
Lansing, MI , USA
JEONGMIN CHOI • Divisions of Plant Science and Biochemistry , University of Missouri ,
Columbia, MO , USA
XUEHUI FENG • Monsanto Company , St. Louis, MO , USA
TIMOTHY E. GOOKIN • Department of Biology, Mueller Laboratory, The Pennsylvania State
University , University Park, PA , USA
DIETER HACKENBERG • Donald Danforth Plant Science Center , St. Louis, MO , USA
PIERS A. HEMSLEY • Division of Plant Sciences, College of Life Sciences, University of
Dundee , Dundee, UK
JIRONG HUANG • National Key Laboratory of Plant Molecular Genetics, Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences , Shanghai, China
INHWAN HWANG • Division of Integrative Biosciences and Biotechnology , Pohang University
of Science and Technology , Pohang , South Korea ; Division of Molecular Life Sciences,
Pohang University of Science and Technology , Pohang , South Korea
ZHAOQING JIN • Biosciences Division, Oak Ridge National Laboratory , Oak Ridge, TN , USA
LON S. KAUFMAN • Molecular, Cell, and Developmental Biology , University of Illinois at
Chicago , Chicago , IL , USA ; Department of Biological Sciences, University of Illinois
at Chicago , Chicago, IL , USA
MYOUNG HUI LEE • Division of Integrative Biosciences and Biotechnology , Pohang
University of Science and Technology , Pohang , South Korea
YONGJIK LEE • Division of Integrative Biosciences and Biotechnology , Pohang University of
Science and Technology , Pohang , South Korea
GUOJING LI • Department of Biology, Hong Kong Baptist University , Kowloon Tong,
Kowloon , Hong Kong
ix
