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METHODS IN M O L E C U L A R B I O L O G Y TM

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For other titles published in this series, go to

www.springer.com/series/7651
Plant Systems Biology

Edited by

Dr. Dmitry A. Belostotsky

School of Biological Sciences,
University of Missouri, Kansas City, MO, USA
Editor
Dmitry A. Belostotsky
School of Biological Sciences
University of Missouri
Kansas City, MO 64110
USA
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-60327-562-0 e-ISBN 978-1-60327-563-7
DOI 10.1007/978-1-60327-563-7
Springer Dordrecht Heidelberg London New York
Library of Congress Control Number: 2009921811

# Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer ScienceþBusiness Media, LLC, 233 Spring Street, New York, NY 10013,
USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as
such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Printed on acid-free paper

Springer is part of Springer ScienceþBusiness Media (www.springer.com)

Dedicated in memory of Dmitry Belostotsky, who passed away
just before the publication of the volume. Special thanks are
extended to Julia Chekanova for her gracious help during the
book’s final weeks.
Foreword
Systems biology has been called many things by many people. Rather than making
another attempt at an all-encompassing definition, it may be better to take an historical
perspective. Back at the dawn of time there was molecular biology, whose goal was to
identify individual genes. With a gene in hand, one then searched upstream and down-
stream for other genes that acted on it or that it targeted. This led to the description of
linear pathways with little arrows between each of the genes. Then came genomics with
its high-throughput technologies to determine the expression of all genes, proteins,
metabolites, etc. The output was usually a long list of cellular components ordered by
expression level or some other metric. These were parsed for meaning based on where
something was found on the list.
What systems biology has brought that is new and different is an emphasis on finding
the connections among the parts. From these connections, the hope is that new
properties will be identified that were not apparent from just staring at the list of
parts. These are called ‘‘emergent properties.’’ But systems biology does not stop
there. After the connections are found and networks begin to emerge, the next step is
to characterize the dynamic properties of these networks. Accomplishing this requires
perturbing the system and then determining how the system responds. In biology,
perturbations can take the form of external stimuli such as sunlight or withholding a
nutrient. They can also be at the level of mutations that alter gene function or
expression.
A distinguishing feature of systems biology is the integration of quantitative
analytical and modeling approaches. In the days of molecular biology, the view of
quantitative analysis was, ‘‘If you have to use statistics, it means you need to do another
experiment.’’ With the advent of genomics, most scientists realized that they needed
help to make sense of the masses of data. Nevertheless, the general approach was that
of a ‘‘hand-off ’’ – the experimental biologist would find someone with quantitative
expertise to ‘‘analyze my data.’’ When the analysis was completed it would be handed
back to the biologist and that was the end of the interaction. The complexity of dynamic
systems has convinced most biologists that the human brain needs mathematical
formalisms to make any sense of the processes being studied. This means that systems
biology is by and large practiced by collaborative teams, which comprise experimental-
ists and theorists, with equal weighting between them.
In this book you will find chapters that describe how to identify cellular components
as well as the interactions among these components. You will also find chapters that
describe methods for perturbing biological systems such as the use of small molecules in
chemical genomics. Fittingly, a large portion of the book is devoted to quantitative
approaches to analyze and model the interactions, emergent properties, and dynamics
of the networks identified.
This work focuses on systems biology applied to plants. For many of the approaches
described here there is no distinction between plants and animals. However, it is

vii
viii Foreword

appropriate to focus an entire book on plant systems biology as plants have been in the
vanguard of this field. The sequencing of the Arabidopsis genome opened the way for a
host of new and innovative approaches to understanding plant biology. From live
imaging of protein dynamics in floral meristems to the ability to follow chromosome
dynamics in individual cells, plant biologists are among the pioneers in this area. No
matter how you choose to define systems biology, it is likely to play an increasingly
important role in elucidating the mysteries of plant biology.

Philip N. Benfey
Preface

Plant Systems Biology: Shooting a Moving Target

Plant systems biology is a fairly new art form. Unsurprisingly, its practitioners come
in a variety of different flavors, and accordingly, there exist a great many conflicting
definitions of what this art form really is (although this probably is true of any art form).
Researchers have been entering this field from all walks of scientific life – there are classical
plant physiologists by training, wet bench gene expression biologists like myself, cell
biologists, mathematicians, statisticians, bioinformaticians, software engineers, and other
more esoteric types ranging all the way to astrophysicists, etc.
The eclectic nature of this proverbial melting pot is also reflected in the content
of this volume, which contains sections covering topics from systems biology of plant
gene expression to analysis of networks, pathways, specific statistical issues and novel
computational tools, imaging-based tools as well as chemical genetic, metabolomic,
and integrative methods that cannot be easily pigeonholed.
While the definition of what plant systems biology really is may still be evolving, its
key leading figures have clearly emerged and who they are is largely beyond dispute.
Indeed, it is quite obvious who is driving the field forward and paving the way for others
who follow in their wake and broaden the path. It is for that reason that the foreword to
this volume is written by Philip Benfey, whose pioneering studies in the field of systems
biology of gene expression have received wide recognition far beyond the plant
community.
While the natural evolution of the field has been rapid and successful, it has become
quite obvious that the time has come for setting up dedicated training programs
in order to sustain this remarkable progress. This is already happening of course, in
the form of IGERT and other training grants, iPlant initiative, etc., but additional
modalities are needed. It is also the hope of the editor that this volume will make a
contribution to achieving this goal as well.
In closing, I would like to acknowledge the contributions of the members of my own
group over the years, and particularly that of Julia Chekanova, as well as the expert
editorial assistance of Teresa Crew, without whom this volume would never have seen
the light of day. Gene expression studies in my lab have been supported by grants from
NSF, USDA, BARD, and NIH.

Dmitry A. Belostotsky

ix
Contents
Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

SECTION I: SYSTEMS BIOLOGY OF PLANT GENE EXPRESSION

1. Gene-Specific and Genome-Wide ChIP Approaches to Study Plant
Transcriptional Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kengo Morohashi, Zidian Xie, and Erich Grotewold
2. Genome-Wide Analysis of RNA–Protein Interactions in Plants . . . . . . . . . . . . . . . 13
Alice Barkan
3. Whole-Genome Microarrays: Applications and Technical Issues. . . . . . . . . . . . . . . 39
Brian D. Gregory and Dmitry A. Belostotsky
4. Manipulating Large-Scale Arabidopsis Microarray Expression Data: Identifying
Dominant Expression Patterns and Biological Process Enrichment . . . . . . . . . . . . 57
David A. Orlando, Siobhan M. Brady, Jeremy D. Koch,
José R. Dinneny, and Philip N. Benfey
5. Applications of Ultra-high-Throughput Sequencing . . . . . . . . . . . . . . . . . . . . . . . 79
Samuel Fox, Sergei Filichkin, and Todd C. Mockler
6. Isolation of Plant Polysomal mRNA by Differential Centrifugation and Ribosome
Immunopurification Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Angelika Mustroph, Piyada Juntawong, and Julia Bailey-Serres
7. Chromatin Charting: Global Mapping of Epigenetic Effects . . . . . . . . . . . . . . . . . 127
Chongyuan Luo and Eric Lam
8. Clone-Based Functional Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Annick Bleys, Mansour Karimi, and Pierre Hilson
SECTION II: NETWORKS, PATHWAYS, STATISTICAL ISSUES, AND NOVEL
COMPUTATIONAL TOOLS
9. Challenges and Approaches to Statistical Design and Inference
in High-Dimensional Investigations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Gary L. Gadbury, Karen A. Garrett, and David B. Allison
10. Discrete Dynamic Modeling with Asynchronous Update, or How to Model
Complex Systems in the Absence of Quantitative Information . . . . . . . . . . . . . . . . 207
Sarah M. Assmann and Réka Albert
11. Quantification of Variation in Expression Networks . . . . . . . . . . . . . . . . . . . . . . . . 227
Daniel J. Kliebenstein
12. Co-expression Analysis of Metabolic Pathways in Plants . . . . . . . . . . . . . . . . . . . . . 247
Ann Loraine
13. Integration of Metabolic Reactions and Gene Regulation . . . . . . . . . . . . . . . . . . . 265
Chen-Hsiang Yeang

xi
xii Contents

14. Applying Word-Based Algorithms: The IMEter . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

Ian F. Korf and Alan B. Rose
SECTION III: IMAGING
15. Live-Imaging and Image Processing of Shoot Apical Meristems of Arabidopsis
thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
G. Venugopala Reddy and A. Roy-Chowdhury
16. Computer Vision as a Tool to Study Plant Development . . . . . . . . . . . . . . . . . . . . 317
Edgar P. Spalding
SECTION IV: CHEMICAL GENETIC, METABOLOMIC, AND INTEGRATIVE
METHODS FOR PLANT SYSTEMS BIOLOGY
17. Metabolomics of Plant Volatiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Anthony V. Qualley and Natalia Dudareva
18. Chemical Genomics Approaches in Plant Biology. . . . . . . . . . . . . . . . . . . . . . . . . . 345
Lorena Norambuena, Natasha V. Raikhel, and Glenn R. Hicks
19. Comparison of Quantitative Metabolite Imaging Tools and Carbon-13
Techniques for Fluxomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
Totte Niittylae, Bhavna Chaudhuri, Uwe Sauer, and Wolf B. Frommer
20. Democratization and Integration of Genomic Profiling Tools . . . . . . . . . . . . . . . . 373
Michael R. Sussman, Edward L. Huttlin, and Dana J. Wohlbach
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 395
Contributors
RÉKA ALBERT • Physics Department, Penn State University, University Park, PA, USA
DAVID B. ALLISON • Section on Statistical Genetics, Department of Biostatistics,
University of Alabama at Birmingham, Birmingham, AL, USA
SARAH M. ASSMANN • Biology Department, Penn State University, University Park,
PA, USA
JULIA BAILEY-SERRES • Center for Plant Cell Biology and Department of Botany and
Plant Sciences, University of California, Riverside, CA, USA
ALICE BARKAN • Institute of Molecular Biology, University of Oregon, Eugene, OR, USA
DMITRY A. BELOSTOTSKY • School of Biological Sciences, University of Missouri Kansas
City, Kansas City, MO, USA
PHILIP N. BENFEY • Department of Biology, Duke University, Durham, NC, USA; IGSP
Center for Systems Biology, Duke University, Durham, NC, USA
ANNICK BLEYS • Department of Plant Systems Biology, Flanders Institute for
Biotechnology (VIB), Gent, Belgium; Department of Molecular Genetics, Ghent
University, Gent, Belgium
SIOBHAN M. BRADY • Department of Biology, Duke University, Durham, NC, USA;
IGSP Center for Systems Biology, Duke University, Durham, NC, USA
BHAVNA CHAUDHURI • Department of Plant Biology, Carnegie Institution for Science,
Stanford, CA, USA
JOSÉ R. DINNENY • Department of Biology, Duke University, Durham, NC, USA; IGSP
Center for Systems Biology, Duke University, Durham, NC, USA
NATALIA DUDAREVA • Department of Horticulture and Landscape Architecture, Purdue
University, West Lafayette, IN, USA
SERGEI FILICHKIN • Department of Botany and Plant Pathology and Center for Genome
Research and Biocomputing, Oregon State University, Corvallis, OR, USA
SAMUEL FOX • Department of Botany and Plant Pathology and Center for Genome
Research and Biocomputing, Oregon State University, Corvallis, OR, USA
WOLF B. FROMMER • Department of Plant Biology, Carnegie Institution for Science,
Stanford, CA, USA
GARY L. GADBURY • Department of Statistics, Kansas State University, Manhattan, KS, USA
KAREN A. GARRETT • Department of Plant Pathology, Kansas State University,
Manhattan, KS, USA
BRIAN D. GREGORY • Plant Biology Laboratory, The Salk Institute for Biological Studies,
La Jolla, CA, USA
ERICH GROTEWOLD • Department of Plant Cellular & Molecular Biology and Plant
Biotechnology Center, The Ohio State University, Columbus, OH, USA
GLENN R. HICKS • Institute for Integrative Genome Biology and Center for Plant
Cell Biology, Department of Botany and Plant Sciences, University of California,
Riverside, CA, USA

xiii
xiv Contributors

PIERRE HILSON • Department of Plant Systems Biology, Flanders Institute for

Biotechnology (VIB), Gent, Belgium; Department of Molecular Genetics, Ghent
University, Gent, Belgium
EDWARD L. HUTTLIN • Department of Biochemistry, University of Wisconsin-Madison,
Madison, WI, USA
PIYADA JUNTAWONG • Center for Plant Cell Biology and Department of Botany and
Plant Sciences, University of California, Riverside, CA, USA
MANSOUR KARIMI • Department of Plant Systems Biology, Flanders Institute for
Biotechnology (VIB), Gent, Belgium; Department of Molecular Genetics, Ghent
University, Gent, Belgium
DANIEL J. KLIEBENSTEIN • Department of Plant Sciences, University of California,
Davis, CA, USA
JEREMY D. KOCH • 3051 Cassel PI, Davis, CA 95616
IAN F. KORF • Genome Center and Section of Molecular and Cellular Biology, University
of California, Davis, CA, USA
ERIC LAM • Biotechnology Center for Agriculture and the Environment, Rutgers
University, New Brunswick, NJ, USA
ANN LORAINE • Bioinformatics Research Center , University of North Carolina-Charlotte,
Kannapolis, NC, USA
CHONGYUAN LUO • Biotechnology Center for Agriculture and the Environment, Rutgers
University, New Brunswick, NJ, USA
TODD C. MOCKLER • Department of Botany and Plant Pathology and Center for
Genome Research and Biocomputing, Oregon State University, Corvallis, OR, USA
KENGO MOROHASHI • Department of Plant Cellular & Molecular Biology and Plant
Biotechnology Center, The Ohio State University, Columbus, OH, USA
ANGELIKA MUSTROPH • Center for Plant Cell Biology and Department of Botany and
Plant Sciences, University of California, Riverside, CA, USA
TOTTE NIITTYLAE • Department of Plant Biology, Carnegie Institution for Science,
Stanford, CA, USA
LORENA NORAMBUENA • Plant Molecular Biology Laboratory, Department of Biology,
Faculty of Sciences, University of Chile, Santiago, Chile
DAVID A. ORLANDO • Department of Biology, Duke University, Durham, NC,USA;
IGSP Center for Systems Biology, Duke University, Durham, NC, USA
ANTHONY V. QUALLEY • Department of Horticulture and Landscape Architecture,
Purdue University, West Lafayette, IN, USA
NATASHA V. RAIKHEL • Institute for Integrative Genome Biology and Center for Plant
Cell Biology, Department of Botany and Plant Sciences, University of California,
Riverside, CA, USA
G. VENUGOPALA REDDY • Department of Botany and Plant Sciences, University of
California, Riverside, CA, USA
ALAN B. ROSE • Section of Molecular and Cellular Biology, University of California,
Davis, CA, USA
A. ROY-CHOWDHURY • Department of Electrical Engineering, University of California,
Riverside, CA, USA
UWE SAUER • Institute for Molecular Systems Biology, ETH Zürich, Zürich, Switzerland
EDGAR P. SPALDING • Department of Botany, University of Wisconsin, Madison, WI, USA
 Contributors xv

MICHAEL R. SUSSMAN • Department of Biochemistry, UW Biotechnology Center,

University of Wisconsin-Madison, Madison, WI, USA
DANA J. WOHLBACH • Department of Genetics, University of Wisconsin-Madison,
Madison, WI, USA
ZIDIAN XIE • Department of Plant Cellular & Molecular Biology and Plant Biotechnology
Center, The Ohio State University, Columbus, OH, USA
CHEN-HSIANG YEANG • Institute of Statistical Science, Academia Sinica, Nankang,
Taipei, Taiwan, R.O.C.
 Chapter 1

Gene-Specific and Genome-Wide ChIP Approaches to Study

Plant Transcriptional Networks
Kengo Morohashi, Zidian Xie, and Erich Grotewold

Abstract
Chromatin immunoprecipitation (ChIP) provides a versatile tool to investigate the in vivo location of
DNA-binding proteins on genomic DNA. ChIP approaches are gaining significance in plants, in cases
when entire genome sequences are available (e.g., Arabidopsis), for which several high-density oligo arrays
have been or are being developed. Nevertheless, plant ChIP and ChIP-chip still present some technical
challenges. Here, we describe general methods for ChIP and ChIP-chip, which have been successfully
applied to maize and Arabidopsis.

Key words: Regulatory network, chromatin immunoprecipitation, transcription factor, histone.

1. Introduction

Protein–DNA interactions are central for life, for example as part

of normal chromatin assembly and in the recognition of specific
cis-regulatory elements (CRE) by transcription factors (TFs).
CREs provide the blueprints for the integration of cellular signals
on the DNA, with the proper gene expression response furnished
by the tethering of sets of TFs to specific DNA motifs and their
interactions with the basal transcription machinery. Understand-
ing which of the thousands of TFs expressed by plant genomes
recognize which CREs and establishing how combinations of TFs
on specific promoters contribute to the regulation of gene expres-
sion pose significant challenges in elucidating the architecture of
plant transcriptional regulatory networks (1).

Dmitry A. Belostotsky (ed.), Plant Systems Biology, vol. 553

ª Humana Press, a part of Springer Science+Business Media, LLC 2009
DOI 10.1007/978-1-60327-563-7_1 Springerprotocols.com

3
4 Morohashi, Xie, and Grotewold

Methods to identify protein–DNA interactions include experi-

mental and computational approaches, or combinations thereof
(1). Experimental approaches involve investigating the formation
of protein–DNA complexes for example by electrophoretic mobi-
lity shift assays (EMSA) or by exploring the specific DNA sequence
recognized by a TF on a given fragment of DNA using chemical or
nuclease footprinting techniques. These techniques, however,
involve in vitro protein–DNA interactions and their application
depends on the availability of a DNA fragment containing the
regulatory sequences. Two main approaches are currently available
to identify and/or validate the direct in vivo targets of a TF. The
first one involves expressing a fusion of the TF to GR (GR corre-
sponds to the hormone-binding domain of the glucocorticoid
receptor) and identifying the mRNAs induced/repressed in the
presence of the GR ligand (dexamethasone, DEX), in the presence
of an inhibitor of translation (e.g., cycloheximide, CHX) (2–6).
The second one involves identifying the DNA sequences that a TF
binds in vivo, using chromatin immunoprecipitation (ChIP)
assays. ChIP not only provides a tool to identify the in vivo loca-
tion of DNA-binding proteins on the DNA but also complements
many of the downfalls of EMSA and footprinting. The experimen-
tal steps necessary for implementing ChIP, or combinations of
ChIP with the hybridization of microarrays (ChIP-chip) corre-
sponding to entire genomes (tiling arrays) or just to the promoter
space of a genome (promoter arrays), are the subject of this
chapter.
In ChIP, intact tissues or cells are treated with a cross-linking
agent that covalently links the protein with the DNA. The chro-
matin is then sheared (using enzymatic or mechanical methods)
and the covalently linked protein–DNA complex is enriched by
immunoprecipitation (IP) using the specific antibodies to the
proteins (7). Multiple cross-linking agents that provide different
spacer lengths are available for ChIP (see Note 1). Formaldehyde
reacts with the amino groups of cytosines, guanines, and adenines
and the imino groups of thymines and guanines on the DNA,
although the reaction with imino groups is likely to be favored in
single-stranded DNA regions. From the protein side, several
amino acids are targeted by formaldehyde, including Lys, Arg,
Trp, and His. The final product of the reaction is the joining
of the two amino groups by a methylene bridge. The uniqueness
of this reaction is that it is reversible in aqueous solutions, and
the reversion of the cross-linking can be significantly increased
by incubation at 65C. The reversibility of the formaldehyde-
mediated reaction furnishes an advantage that has made it the
favorite reagent for cross-linking. It should be noted, however,
that in some instances it is worth considering other cross-linking
agents, if the desired results are not obtained with formalde-
hyde (8).
 Plant ChIP Technique 5

ChIP-chip (aka ChIP-on-chip or genome-wide location

analysis) involves the hybridization of a microarray representing a
fraction or the entire genome space with the DNA resulting from
the ChIP experiment (9). In plants, several arrays have become
available over the past couple of years for ChIP-chip experiments;
for example, for Arabidopsis, promoter (10, 11) and complete
tiling genome (10, 12, 13) arrays are available. In most instances,
the ChIPed DNA needs to be amplified prior to microarray hybri-
dization, because nanogram quantities of DNA are precipitated,
for example when using antibodies against a specific TF. Several
different amplification methods are available (14), and some are
discussed later. A plethora of statistical approaches have been
developed for the analysis of ChIP-chip results [e.g., (9, 15)];
their discussion and application are however beyond the scope
of this chapter. ChIPed DNA, however, can also be analyzed
by methods other than the hybridization to a microarray. For
example, the ChIP-Paired End diTag (PET) method results in
the generation of short sequence tags from the enriched target
DNA after a ChIP experiment (16). Alternatively, the ChIPed
DNA can be cloned and sequenced [e.g., (17)].
ChIP approaches are gaining significance in plants, parti-
cularly for those for which entire genome sequences are avail-
able (e.g., Arabidopsis). While ChIP-chip experiments have
been performed on just a handful of Arabidopsis TFs including
HY5 (11) and TGA2 (10), ChIP is becoming an increasingly
popular method to validate in vivo TF–DNA interactions pre-
dicted by other methods. Moreover, ChIP-chip can be applied
to identify the epigenetic control of the transcriptional
regulation.

2. Materials

1. Salmon sperm/protein A-agarose (Upstate P/N 16-157)

2. PCI: phenol:chloroform:isoamylic alcohol (25:24:1)
3. Buffer A: 0.4 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA,
1 mM PMSF (see Note 2), 1% formaldehyde
4. Lysis buffer: 50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1%
SDS, 10 mM Na butyrate, 1 mM PMSF, 1X plant proteinase
inhibitor cocktail (Sigma) (see Note 2)
5. LNDET: 0.25 M LiCl, 1% NP40, 1% sodium deoxycholate,
1 mM EDTA
6. Elution buffer: 1% SDS, 0.1 M NaHCO3, 0.25 mg/ml
proteinase K
6 Morohashi, Xie, and Grotewold

7. PCR purification kit (QIAGEN), DNA Clean &

Concentrator – 25 (Zymo Research)
8. GenomePlex Whole Genome Amplification kit (Sigma, P/N
WGA-1): 10X library buffer, 10X library stabilization solu-
tion, library preparation enzyme
9. GeneChip1 Arabidopsis Tiling 1.0R Array (Affymetrix)

3. Methods

3.1. Chromatin 1. Immerse tissue into buffer A in a 50 ml falcon tube and keep it
Immunoprecipitation under vacuum for 20 min (see Notes 3 and 4).
(ChIP) in Plants 2. Add 2 M glycine to a final concentration of 0.1 M and con-
tinue vacuum for 10 min.
3.1.1. Cross-Linking
3. Wash the tissue with excess amount of distilled water and
Proteins to DNA
remove as much water as possible.
4. Grind tissue in liquid nitrogen and resuspend in 400 ml of lysis
buffer (see Notes 5 and 6).

3.1.2. Sonication of 1. Shear DNA by sonication to a fragment length that ranges

Chromatin (see Note 7) between 100 bp and 1000 bp (500 bp on average) in an
eppendorf tube (see Notes 8 and 9).
2. Centrifuge at 10,000  g for 10 min at 4C.

3.1.3. Immunoprecipitation 1. Pre-clear supernatant with 30 ml of salmon sperm/protein

(see Note 10) A-agarose for at least 60 min with rotation at 4C.
2. Transfer 100 ml of supernatant into three new eppendorf
tubes and add the antibodies (see Notes 11 and 12). Keep
approximately 100 ml of extract as the input fraction.
3. Incubate overnight with rotation at 4C.
4. Add 30 ml of salmon sperm/protein A-agarose slurry and
continue incubation with rotation at 4C for at least 2 h.
5. Centrifuge at 750  g (3000 rpm for microcentrifuge) for
1 min at 4C.

3.1.4. Washes 1. Add 0.5 ml of lysis buffer, invert six times, centrifuge at
750  g for 1 min, and discard supernatant (see Note 12).
2. Add 0.5 ml of lysis buffer, rotate for 5 min, centrifuge at
750  g for 1 min, and discard supernatant.
3. Add 0.5 ml of LNDET, invert six times, centrifuge at 750  g
for 1 min, and discard supernatant.
4. Add 0.5 ml of LNDET, rotate for 5 min, centrifuge at 750  g
for 1 min, and discard supernatant.
 Plant ChIP Technique 7

5. Add 0.5 ml of TE, invert six times, centrifuge at 750  g for

1 min, and discard supernatant.
6. Add 0.5 ml of TE, rotate for 5 min, centrifuge at 750  g for
1 min, and discard supernatant.

3.1.5. Reverse Cross- 1. Add 40 ml of elution buffer and incubate at 65C for 15 min.
Linking 2. Centrifuge at 750  g for 1 min and transfer supernatant to
new tube.
3. Repeat eluting steps. The final elution volume should be now
80 ml. In parallel, add 70 ml of elution buffer into 10 ml of
input fraction for the input control, which represents 10% of
the cross-linked DNA (see Note 13).
4. Incubate all samples overnight at 65C.

3.1.6. DNA Isolation Extract the DNA by using the PCR purification kit (QIAGEN). Elute
in 30 ml of EB buffer (10 mM Tris–HCl, pH 8.5) (see Note 14).

3.1.7. Quantification of We generally use 1 ml of eluted DNA samples for standard PCR
ChIPed DNA (see Note 15) and normalization (see Note 16), although larger
quantities can be used, if necessary.

3.2. ChIP-chip 1. Add 1 ml of 10X fragmentation buffer to 10 ml ChIPed DNA

solution.
3.2.1. DNA Amplification 2. Place the tube in a thermal cycler at 95C for exactly 4 min
After ChIP (see Note 17) (see Note 18).
3. Immediately cool the sample on ice and then centrifuge
briefly.
4. Add 2 ml 1X library buffer to 11 ml material (see Note 19).
5. Add 1 ml library stabilization solution. Mix by pipetting. Place
at 95C for 2 min in thermal cycler.
6. Add 1 ml library preparation enzyme. Mix by pipetting.
7. Incubate in thermal cycler as follows:
16C for 20 min24C for 20 min
37C for 20 min
75C for 5 min
4C hold
8. Add the following reagents into the library-prepared sample:
7.5 ml of 10X Amplification Master Mix
47.5 ml nuclease-free H2O
5 ml WGA DNA polymerase
9. Incubate in thermal cycler using the following program:
95C for 3 min, then 14 cycles of
94C for 15 s
8 Morohashi, Xie, and Grotewold

65C for 5 min, then

4C hold
10. Purify the sample using the DNA Clean & Concentrator – 25
system.
11. Quantify the amount of DNA by A260. If the total DNA amount
is less than 1 mg, reamplify the sample using GenomePlex WGA
Reamplification kit starting from step 1.
12. Use 5–10 mg of amplified DNA for the hybridization of the
array (see Note 20).

3.2.2. DNA Fragmentation, For DNA fragmentation, labeling, hybridization, wash, and detection,
Labeling, Tiling Array we follow the Affymetrix 100K protocol (http://www.affymetrix.
Hybridization, Wash, and com/support/technical/byproduct.affx?product=100k).
Detection

3.2.3. Data Analysis The complete information on the Affymetrix tiling array is pro-
(see Notes 21 and 22) vided by the .CEL file. To analyze the data, several tools are
currently available, with MAT (model-based analysis of tiling
array) providing a convenient first step (15). To use MAT, a
UNIX platform or equivalent is required. MAT requires the .CEL,
.bpmap, and .lib files. The .CEL file contains the signals of all the
probes on the array, the .bpmap files provide information on the
probe locations and copy numbers, and the .lib file contains the
repeat information.

4. Notes
1. These include formaldehyde, dimethyl adipimidate (DMA),
dimethyl pimelimidate (DMP) dimethyl suberimidate (DMS),
N-hydroxysuccinimide (NHS), tris-succinimidyl aminotriace-
tate (TSAT), disuccinimidyl suberate (DSS), disuccinimidyl
glutarate (DSG), and ethylene glycol bis(succinimidylsuccinate)
(EGS) (8, 18).
2. PMSF, which is unstable in aqueous solution, and the protei-
nase inhibitor are added just before use.
3. In general, we use approximately 240 mg tissue for three
precipitations plus input, which consist of IgG for the nega-
tive control, histone 3 (H3) antibody for the positive control,
the antibody against the specific TF or tag and input. Alter-
natively 1.2 g of tissue for three precipitations might be used
in a large-scale experiment, if the small-scale experiment does
not yield enough DNA.
4. We have used various tissues so far including Arabidopsis and
maize seedlings, Arabidopsis and maize leaf tissues, Arabidopsis
root tissue, Arabidopsis flower buds, maize Black Mexican
 Plant ChIP Technique 9

Sweet cells, and maize protoplasts transiently expressing

epitope-tagged transcription factors. If not used immediately,
cross-linked samples can be stored at –80C.
5. The quality of grinding is very critical for the successful out-
come of the experiments and the tissues must be ground very
well.
6. We also have homogenized tissues in microcentrifuge tubes
using a small plastic pestle in lysis buffer, in cases when only
small quantities of tissue were available. The quality of the
small-scale homogenization is as good as grinding larger
quantities of tissue in liquid nitrogen, yet care must be used
in not warming the extract more than necessary when holding
the tube between the finger tips.
7. Alternatively, methods are available that use DNase I nuclease.
8. Sonication is the most critical step for the success of ChIP
experiments. The ideal sonication conditions depend on a
number of factors including the volume of extract, the soni-
cator tip size, and the sonicator itself. An optimal sonication
condition should be identified prior to performing the ChIP
experiment. For example, when using Arabidopsis seedling
we have determined that in a Vibra Cell Sonicator (Sonics&
Materials) five repeats of 15 s each at 10% amplitude provide
the best results. To determine the optimal sonication con-
ditions, various parameters should be tested such as the
amplitude and duration of the sonication cycles (e.g., 5,
10, 40% of amplitude for 0, 10, 30, 60, 300 s) using cross-
linked extract. Then, after reverse cross-linking and DNA
purification, the size of the fragmented DNA is verified by
electrophoresis.
9. Avoid making bubbles during sonication. Bubbles cause a
significant reduction in sonication efficiency.
10. ChIP results strongly depend on the quality (affinity and
specificity) of the antibody. We use antibodies against his-
tones as experimental positive controls since commercially
available antibodies that recognize a number of histone-tail
modifications have been extensively used in ChIP experiment
in plants, yeast, and animals.
11. We succeeded in obtaining reproducible signals by using
antibodies that recognize acetylated H3 at position K9
(H3K9ac) (Upstate P/N 06-599) and anti-GFP (abcam P/N
ab290). Antibody amounts are variable (19–21). For exam-
ple, we use 2 mg of IgG, 1 mg of H3K9ac, and 1 ml of anti-
GFP antibodies for 100 ml of extract. However, monoclonal
anti-myc epitope antibodies (line 9E10) have so far resulted
in faint and irreproducible signals when using Arabidopsis
extracts.
10 Morohashi, Xie, and Grotewold

12. The washing steps should be ideally performed in the cold

room.
13. Make sure that the final concentration of SDS, NaHCO3, and
proteinase K in the input sample is the same as in the other
samples when adjusting the volume.
14. Elution volume depends on the amount of starting tissue. For
example, if we start with 200 mg of plant tissue, we elute in
30 ml. For the isolation of DNA from the input extract, PCI
extraction can be used when starting from large quantities of
plant material. The purification using the QIAGEN columns
is performed after the PCI extraction.
15. Since the ChIPed DNA is usually in very low amounts, the
detection of the target DNA requires PCR. Therefore, there
is a risk of PCR amplification bias; thus we strongly recom-
mend quantitative PCR or semi-quantitative PCR to compare
the enrichment of the target DNA with respect to the input
control.
16. To accurately compare the quantity of ChIPed and input
DNA, we recommend a double normalization using input
DNA and a reference primer set. The ratio between the
input and ChIPed DNA is a good index for the enrichment
during the ChIP. However, it is always possible that there is a
bias, for example because of non-specific binding of DNA to
the beads. To rule out such artifacts, the reference primer set
is used. The reference primer set should not to be a target of
the TF in study, of course. For Arabidopsis, for example, we
routinely use primers corresponding to ACT2/7 (15). The
final normalization can then be done using the following
formula:
ðChIPedDNAtarget =InputDNAtarget Þ
ðChIPedDNAreference =InputDNAreference Þ

17. Amplification is one of the most critical steps for ChIP-chip.

Among several available amplification methods, we use the
GenomePlex Whole Genome Amplification (WGA) kit
(Sigma) with the modifications previously described (14).
Using WGA, we have successfully obtained reproducible results
with various tissues and mutants of Arabidopsis.
18. The incubation time is very critical.
19. The amount of ChIPed DNA is usually less than 1 ng/ml; thus
accurately measuring the amount of DNA is challenging. We
generally use 11 ml as start material for the amplification.
20. For the purpose of this manuscript, we are primarily referring
to the Affymetrix GeneChip1 Arabidopsis Tiling 1.0R Array.
We have also used less than 5 mg of amplified DNA, yet the
results have been variable.
 Plant ChIP Technique 11

21. The ideal way to perform a ChIP-chip experiment is by the

side-by-side comparison of wild-type and mutant tissue, the
latter corresponding to tissues lacking the specific TF. In such
a case, the ChIP-chip analysis is performed by comparing the
input and ChIP DNA from both the wild-type and mutant
samples. Alternatively, if using plants expressing an epitope-
tagged version of the TF, plants not expressing the transgene
can be used as the mutant sample.
22. There are many methods to analyze ChIP-chip data. Gener-
ally, the analysis can be divided into two steps: normalization
and detection of enriched signal region. The MAT algorithm
takes care of both steps at the same time. It standardizes the
probe value through the probe model, which consist of base-
line probe behavior by considering probe sequence and copy
number. Therefore, it eliminates the normalization step.

Acknowledgments

We thank Herbert Auer for helping us develop the ChIP-chip

method for Arabidopsis and for comments on this manuscript,
Manli Davis for helping us grow and collect plant materials, and
Marko Djordjevic for assistance with the analysis of ChIP-chip
data. This research was supported by National Science Foundation
(NSF) grant MCB-0418891 and by NRI grant 2007-35318-
17805 from the USDA-CSREES to E.G.

References

1. Grotewold, E. and Springer, N. (2009) 5. Wang, D., Amornsiripanitch, N., and Dong,
Decoding the transcriptional hardwiring X. (2006) A genomic approach to identify
of the plant genome. In: Coruzzi, G. and regulatory nodes in the transcriptional net-
Gutierrez, R. (eds). In Systems Biology. work of systemic acquired resistance in
Blackwell Publishing. In Press. plants. PLoS Pathog. 2, e123.
2. Sablowski, R.W. and Meyerowitz, E.M. 6. Wellmer, F., Alves-Ferreira, M., Dubois, A.,
(1998) A homolog of NO APICAL MERIS- Riechmann, J.L., and Meyerowitz, E.M.
TEM is an immediate target of the floral (2006) Genome-wide analysis of gene
homeotic genes APETALA3/PISTILLATA. expression during early Arabidopsis flower
Cell. 92, 93–103. development. PLoS Genet. 2, e117.
3. Spelt, C., Quattrocchio, F., Mol, J., and 7. Wells, J. and Farnham, P.J. (2002) Char-
Koes, R. (2002) ANTHOCYANIN1 of pet- acterizing transcription factor binding
unia controls pigment synthesis, vacuolar sites using formaldehyde crosslinking and
pH, and seed coat development by geneti- immunoprecipitation. Methods. 26,
cally distinct mechanisms. Plant Cell. 14, 48–56.
2121–2135. 8. Nowack, D.E., Tian, B., and Brasier, A.R.
4. Shin, B., Choi, G., Yi, H., et al. (2002) (2005) Two-step cross-linking method for
AtMYB21, a gene encoding a flower-specific identification of NF-KB gene network by
transcription factor, is regulated by COP1. chromatin immunoprecipitation. Biotechni-
Plant J. 30, 23–32. ques. 39, 715–725.
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9. Buck, M.J., Nobel, A.B., and Lieb, J.D. 15. Johnson, W.E., Li, W., Meyer, C.A., et al.
(2005) ChIPOTle: a user-friendly tool for (2006) Model-based analysis of tiling-arrays
the analysis of ChIP-chip data. Genome Biol. for ChIP-chip. Proc. Natl. Acad. Sci. USA.
6, R97. 103, 12457–12462.
10. Thibaud-Nissen, F., Wu, H., Richmond, T., 16. Wei, C.L., Wu, Q., Vega, V.B., et al. (2006)
et al. (2006) Development of Arabidopsis A global map of p53 transcription-factor
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the TGA2 transcription factor in salicylic 17. Denissov, S., van Driel, M., Voit, R., et al.
acid-treated plants. Plant J. 47, 152–162. (2007) Identification of novel functional
11. Lee, J., He, K., Stolc, V., et al. (2007) TBP-binding sites and general factor reper-
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mic binding sites revealed its hierarchical 18. Hermanson, G.T. (1996) Bioconjugate
role in light regulation of development. Techniques. San Diego: Academic Press.
Plant Cell. 19, 731–749. 19. Morohashi, K. and Grotewold, E. (2009) A
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 Chapter 2

Genome-Wide Analysis of RNA–Protein Interactions in Plants

Alice Barkan

Abstract
RNA–protein interactions profoundly impact organismal development and function through their con-
tributions to the basal gene expression machineries and their regulation of post-transcriptional processes.
The repertoire of predicted RNA binding proteins (RBPs) in plants is particularly large, suggesting that the
RNA–protein interactome in plants may be more complex and dynamic even than that in metazoa. To
dissect RNA–protein interaction networks, it is necessary to identify the RNAs with which each RBP
interacts and to determine how those interactions influence RNA fate and downstream processes. Identi-
fication of the native RNA ligands of RBPs remains a challenge, but several high-throughput methods for
the analysis of RNAs that copurify with specific RBPs from cell extract have been reported recently. This
chapter reviews approaches for defining the native RNA ligands of RBPs on a genome-wide scale and
provides a protocol for a method that has been used to this end for RBPs that localize to the chloroplast.

Key words: RNA–protein interaction, RIP-chip, RNA coimmunoprecipitation, microarray, RNA

binding protein.

1. Introduction

Organismal development, homeostasis, and environmental adap-

tation require the regulated expression of large sets of genes. The
rates of an array of post-transcriptional events are superimposed
upon the transcription rate to determine the output of each gene,
and in some cases, post-transcriptional steps play a dominant role.
Thus, RNA binding proteins (RBPs) that influence the processing,
nuclear export, stability, or translation of RNA subsets are likely to
contribute to the large-scale coordination of gene expression
(reviewed by (1, 2)). RBPs are also at the core of the machineries

Dmitry A. Belostotsky (ed.), Plant Systems Biology, vol. 553

ª Humana Press, a part of Springer Science+Business Media, LLC 2009
DOI 10.1007/978-1-60327-563-7_2 Springerprotocols.com

13
14 Barkan

that localize specific mRNAs within cells, thereby influencing the

localization of protein synthesis to specific subcellular domains in
plants, animals, and fungi (reviewed in (3, 4)).
It has long been appreciated that post-transcriptional mechan-
isms play a major role in determining gene expression levels in
plant mitochondria and chloroplasts (reviewed in (5, 6–9)).
Recently, the importance of post-transcriptional events in dictat-
ing other plant traits has been highlighted by the recovery of genes
encoding nuclear/cytosolic RBPs and microRNAs in genetic
screens for phenotypes affecting diverse processes such as flo-
wering, circadian control, and hormone responses (reviewed in
(10, 11–13)). Genome-wide assays to explore the impact of post-
transcriptional regulatory mechanisms in plants have only recently
begun, but the results thus far suggest that their impact is con-
siderable. For example, the stabilities of mRNA subsets are under
circadian control (14) and stress-induced changes in the transla-
tion of large sets of plant mRNAs have been reported (15, 16).
Although large-scale analyses of regulated changes in splice iso-
form populations have not yet been reported in plants, there is
evidence that both biotic and abiotic stresses influence the alter-
native splicing of plant pre-mRNAs (reviewed in (17)) (18, 19).
Interactions between RNAs and RBPs in ribonucleoprotein
particles (RNPs) underlie the biogenesis, localization, translation,
and turnover of mRNAs, the biogenesis of non-coding RNAs, and
the regulation of all of these processes (20). However, even in the
most intensively studied fungal and animal systems, the functions
and RNA ligands for the vast majority of predicted RBPs remain
unknown (1). The challenge is still greater in plants, whose reper-
toire of predicted RBPs is considerably larger than that in metazoa
(10, 21–26). Gene families encoding homologs of proteins impli-
cated in nuclear pre-mRNA splicing, polyadenylation, and mRNA
decay are expanded in plants (21–23). Additional complexity is
introduced by the maintenance of a third genetic compartment in
plants, the chloroplast, where RNA editing, group I and group II
intron splicing, endonucleolytic mRNA processing, and regulated
translation and mRNA turnover are prevalent (reviewed in (6, 8,
9)). Furthermore, RNA metabolism in plant mitochondria is sub-
stantially more complex than that in metazoa, sharing many fea-
tures with that in chloroplasts (reviewed in (5, 7, 8)). Indeed,
the expansion of two RBP families specifically in the plant lineage
(the CRM and PPR families) appears to be linked to the prominent
roles of post-transcriptional aspects of gene expression in these
organelles (24, 25, 27).
Identification of the RNAs with which each RBP is associated is
at the core of understanding RNP interaction networks. The phe-
notypes conditioned by loss-of-function mutations in RBP genes
can provide clues about their RNA ligands, especially when coupled
with genome-wide assays for changes in mRNA profiles (see, for
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