Table Of ContentM M B TM
ETHODS IN OLECULAR IOLOGY
SeriesEditor
JohnM.Walker
School ofLife Sciences
University ofHertfordshire
Hatfield, Hertfordshire,AL109AB,UK
For further volumes:
http://www.springer.com/series/7651
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Plant Signalling Networks
Methods and Protocols
Edited by
Zhi-Yong Wang
Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, USA
Zhenbiao Yang
Department of Botany and Plant Science, University of California, Riverside, CA, USA
Editors
Zhi-YongWang ZhenbiaoYang
DepartmentofPlantBiology DepartmentofBotanyandPlantScience
CarnegieInstitutionforScience UniversityofCalifornia
Stanford,CA,USA Riverside,CA,USA
ISSN1064-3745 e-ISSN1940-6029
ISBN978-1-61779-808-5 e-ISBN978-1-61779-809-2
DOI10.1007/978-1-61779-809-2
SpringerNewYorkDordrechtHeidelbergLondon
LibraryofCongressControlNumber:2012936833
ªSpringerScience+BusinessMedia,LLC2012
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Preface
Signal transduction is the fundamental mechanism for regulation of cellular activities by
environmentalcuesandregulatorysignals.Signaltransductionisparticularlyimportantfor
plants, whose survival requires proper physiological and developmental responses to the
environmental changes. Genome sequencing has revealed expansion of gene families
encoding signal transduction proteins in plants compared to animals. Genetic studies in
the last two decades have identified receptors and key signal transduction components of
many signaling pathways in plants, mostly in the model system Arabidopsis. However,
plant signaling systems are complex and require diverse approaches and techniques to
dissect. Conceptually, signal transduction involves signal perception by receptors and
activation of receptor activity, intracellular signal relay, which often involves protein–
protein interaction and posttranslational protein modifications such as phosphorylation,
glycosylation, ubiquitination, and oxidation, and regulation of gene expression. Much
progress has been made recently in the plant signal transduction research field thanks to
the development of diverse techniques, including proteomics and mass spectrometry
methods for studying protein modification, biochemical and cell biological tools for
studying protein–protein interactions, genomic techniques for dissecting protein–DNA
interactionandtranscriptionnetworks,andcomputationmethodsthatintegratemolecular
networksintoplantdevelopmentalprocesses.PlantSignalingNetworksdescribesmanyof
theseadvancedresearchmethods.
Chapters1–3describemassspectrometrymethodsforstudyingproteinphosphorylation
and glycosylation. Oneofthemost important classesofplant receptors is thereceptor-like
kinaseslocalizedonthecellsurface.Methodsforanalysisofreceptorkinasephosphorylation
usingmassspectrometryareprovidedinChap.1.Thesemethodshaveyieldedinsightsinto
the molecular details of receptor kinase activation by autophosphorylation and transpho-
sphorylationinreceptorkinasecomplexes.Chapter2describesquantitativemeasurementof
protein phosphorylationin complexsamples, whichis usefulin identifyingphosphorylated
components in signal transduction pathways. Chapter 3 describes enrichment and mass
spectrometryanalysisofO-GlcNAcmodificationofproteins,whichisanimportantprotein
modificationforsignaling.Chapters4–6describeadvancedtwo-dimensionalelectrophoresis
methodsforquantitativeproteomicanalysisofproteinslocalizedontheplasmamembrane,or
modifiedbyphosphorylationorredox.
Genetic approaches are powerful for identifying essential signaling components, but
havelimitationsduetogeneticredundancy.Severalelaboratestrategieshavebeenshownto
be effective in overcoming genetic redundancy. Chapters 7 and 8 describe chemical
genetics—use of small molecule chemicals, to dissect signaling pathways, and Chapter 9
describesanimprovedtoolforgeneratingoverexpressionmutants.
G-proteinsareimportantcomponentsofmanysignaltransductionpathways.Chapters
10 and 11 describe biochemical and cell biological methods for analyzing G-protein
activation. Ubiquitination is another universal mechanism used widely in all cellular
regulatory processes. Specific interactions between E3 ubiquitin ligases and substrate
proteins are key for regulating degradation/accumulation of signaling proteins. Chapters
12and13describeinvivoandinvitromethodsforanalyzingE3–substrateinteractionand
ubiquitination.
v
vi Preface
Most signal transduction pathways regulate development and physiology by
controlling gene expression. Identification of all target genes of a signaling pathway is
key for understanding not only the functions of the pathway but also the regulatory
network that integrates multiple pathways. Chapter 14 describes the use of chromatin
immunoprecipitationfollowedbymicroarray(ChIP-chip)orhigh-throughputsequencing
(ChIP-Seq) for identifying target genes of transcription factors in both Arabidopsis and
rice. Quantitative analysis of gene expression as output of signal transduction provides
effective assays for functions of signaling components. Chapter 15 describes a smart
pooling approach that improves the efficiency of RNA profiling experiments. Chapter 16
describes the powerful cell-based transient gene expression assay for testing functions of
and delineating relationships among signaling components. Chapter 17 describes a
method for profiling un-capped RNA, which can reveal posttranscriptional regulation of
RNA abundance. Finally, Chap. 18 provides brief account of recently developed imaging
andcomputationmethodsforanalyzingbothlocalandglobalpatternsofgeneexpression
andgrowthinArabidopsisshootapicalmeristems(SAMs).
Plant Signalling Networks provides detailed protocols for a wide range of research
approachesincludinggenetics,proteomics,biochemical,cellbiological,andcomputational
approaches. These are powerful methods for understanding various aspects of signaling
networks in plants. We hope that this timely overview of diverse approaches for studying
signaltransductionsystemsprovidesaguideforresearcherstogaincomprehensiveunder-
standingofcomplexsignalingnetworksinplants.
Stanford,CA,USA Zhi-YongWang
Riverside,CA,USA ZhenbiaoYang
Contents
Preface.................................................................... v
Contributors............................................................... ix
1 ExperimentalAnalysisofReceptorKinasePhosphorylation ................... 1
SrijeetK.Mitra,MichaelB.Goshe,andStevenD.Clouse
2 QuantitativeMeasurementofPhosphopeptidesandProteinsvia
StableIsotopeLabelinginArabidopsisandFunctional
PhosphoproteomicStrategies ............................................ 17
NingLi
3 IdentificationofO-linkedb-D-N-acetylglucosamine-Modified
ProteinsfromArabidopsis................................................ 33
Shou-LingXu,RobertJ.Chalkley,Zhi-YongWang,
andAlmaL.Burlingame
4 QuantitativeAnalysisofProteinPhosphorylationUsingTwo-Dimensional
DifferenceGelElectrophoresis ........................................... 47
ZhipingDeng,ShuoleiBu,andZhi-YongWang
5 QuantitativeAnalysisofPlasmaMembraneProteomeUsingTwo-Dimensional
DifferenceGelElectrophoresis ........................................... 67
WenqiangTang
6 IdentificationandVerificationofRedox-SensitiveProteins
inArabidopsisthaliana.................................................. 83
HaiWang,ShengbingWang,andYijiXia
7 Small-MoleculeDissectionofBrassinosteroidSignaling ...................... 95
Mirela-CorinaCodreanu,DominiqueAudenaert,LongNguyen,
TomBeeckman,andEugeniaRussinova
8 AChemicalGeneticsMethodtoUncoverSmallMoleculesforDissecting
theMechanismofABAResponsesinArabidopsisSeedGermination............ 107
YangZhao
9 ActivationTagging ..................................................... 117
XiaopingGouandJiaLi
10 RhoGTPaseActivityAnalysisinPlantCells ................................ 135
TongdaXu
11 AnalysisofInVivoROPGTPaseActivityattheSubcellularLevel
byFluorescenceResonanceEnergyTransferMicroscopy ..................... 145
LeiZhuandYingFu
12 InVivoUbiquitinationAssaybyAgroinfiltration............................ 153
LijingLiu,QingzhenZhao,andQiXie
13 InVitroProteinUbiquitinationAssay..................................... 163
QingzhenZhao,LijingLiu,andQiXie
vii
viii Contents
14 Genome-WideIdentificationofTranscriptionFactor-BindingSites
inPlantsUsingChromatinImmunoprecipitationFollowedbyMicroarray
(ChIP-chip)orSequencing(ChIP-seq).................................... 173
Jia-YingZhu,YuSun,andZhi-YongWang
15 SmartPoolingofmRNASamplesforEfficientTranscriptProfiling............. 189
RaghunandanM.Kainkaryam,AngelaBruex,PeterJ.Woolf,
andJohnSchiefelbein
16 TransientExpressionAssaysforQuantifyingSignalingOutput ................ 195
YajieNiuandJenSheen
17 Genome-WideProfilingofUncappedmRNA............................... 207
YulingJiaoandJose´ LuisRiechmann
18 ComputationalToolsforQuantitativeAnalysisofCellGrowthPatterns
andMorphogenesisinActivelyDevelopingPlantStemCellNiches ............ 217
AnirbanChakraborty,RamKishorYadav,MinLiu,MosesTataw,
KatyaMkrtchyan,AmitRoyChowdhury,andG.VenugopalaReddy
Index..................................................................... 229
Contributors
DOMINIQUE AUDENAERT (cid:1) Department of Plant Systems Biology, VIB,
Ghent, Belgium; Department of Plant Biotechnology and Bioinformatics,
Ghent University, Ghent, Belgium
TOM BEECKMAN (cid:1) Department of Plant Systems Biology, VIB, Ghent, Belgium;
Department of Plant Biotechnology and Bioinformatics, Ghent University,
Ghent, Belgium
ANGELA BRUEX (cid:1) Department of Molecular, Cellular, and Developmental Biology,
University of Michigan, Ann Arbor, MI, USA
SHUOLEI BU (cid:1) Department of Plant Biology, Carnegie Institution for Science,
Stanford, CA, USA
ALMA L. BURLINGAME (cid:1) Department of Pharmaceutical Chemistry,
University of California, San Francisco, CA, USA
ANIRBAN CHAKRABORTY (cid:1) Department of Electrical Engineering,
University of California, Riverside, CA, USA
ROBERTJ. CHALKLEY (cid:1) Department of Pharmaceutical Chemistry,
University of California, San Francisco, CA, USA
AMIT ROY CHOWDHURY (cid:1) Department of Electrical Engineering,
University of California, Riverside, CA, USA
STEVEN D. CLOUSE (cid:1) Department of Horticultural Science,
North Carolina State University, Raleigh, NC, USA
MIRELA-CORINA CODREANU (cid:1) Department of Plant Systems Biology, VIB,
Ghent, Belgium; Department of Plant Biotechnology and Bioinformatics,
Ghent University, Ghent, Belgium
ZHIPING DENG (cid:1) Department of Plant Biology, Carnegie Institution for Science,
Stanford, CA, USA; Institute of Virology and Biotechnology, Zhejiang Academy
of Agricultural Science, Hangzhou, China
YING FU (cid:1) State Key Laboratory of Plant Physiology and Biochemistry,
Department of Plant Sciences, College of Biological Sciences,
China Agricultural University, Beijing, China
MICHAEL B. GOSHE (cid:1) Department of Molecular and Structural Biochemistry,
North Carolina State University, Raleigh, NC, USA
XIAOPING GOU (cid:1) School of life sciences, Lanzhou University, Lanzhou, China
YULING JIAO (cid:1) State Key Laboratory of Plant Genomics, Institute of Genetics and
Developmental Biology, Chinese Academy of Sciences, Chaoyang, Beijing, China
RAGHUNANDAN M. KAINKARYAM (cid:1) Department of Chemical Engineering,
University of Michigan, Ann Arbor, MI, USA; Procter & Gamble Company,
Cincinnati, OH, USA
JIA LI (cid:1) School of life sciences, Lanzhou University, Lanzhou, China
NING LI (cid:1) Division of life science, The Hong Kong University of Science
and Technology, Clear Water Bay, Hong Kong, SAR, China
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