Methods in Molecular Biology 2200 Jose J. Sanchez-Serrano Julio Salinas Editors Arabidopsis Protocols Fourth Edition M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Arabidopsis Protocols Fourth Edition Edited by Jose J. Sanchez-Serrano Centro Nacional de Biotecnología, CSIC Centro Nacional de Biotecnología, Madrid, Spain Julio Salinas Centro de Investigaciones Biologicas, CSIC Madrid Centro de Investigaciones Biologicas, Madrid, Spain Editors JoseJ.Sanchez-Serrano JulioSalinas CentroNacionaldeBiotecnolog´ıa CentrodeInvestigacionesBiologicas CSICCentroNacionalde CSICMadridCentrodeInvestigacionesBiologicas Biotecnolog´ıa Madrid,Spain Madrid,Spain ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-0879-1 ISBN978-1-0716-0880-7 (eBook) https://doi.org/10.1007/978-1-0716-0880-7 ©SpringerScience+BusinessMedia,LLC,partofSpringerNature2021 Thisworkissubjecttocopyright.AllrightsarereservedbythePublisher,whetherthewholeorpartofthematerialis concerned,specificallytherightsoftranslation,reprinting,reuseofillustrations,recitation,broadcasting,reproduction onmicrofilmsorinanyotherphysicalway,andtransmissionorinformationstorageandretrieval,electronicadaptation, computersoftware,orbysimilarordissimilarmethodologynowknownorhereafterdeveloped. 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Preface Itis6yearssincethethirdeditionoftheArabidopsisProtocolsbookappeared.Duringthis time, the field of Plant Molecular Biology has expanded remarkably as reflected by the increasing amount of plant science articles published in the most prestigious general jour- nals. This expansion has been due, not to a small degree, to the development of novel experimentalprocedures,whichnowallowintransigentplantmaterialtobeexaminedwitha similar degree of sophistication as bacteria, yeasts, or animal cells. Relevant also for such expansion has been the use of Arabidopsis thaliana, which continues to be the most important model system for plant scientists. The mature Arabidopsis research community has developed a large array of new methodologies and biological tools, leading to the generation of a vast amount of original data that have significantly contributed to a better understanding of how plants grow, develop, and reproduce in their environments. At this stage, we considered that a new edition of the book could be useful both to update some protocolsalreadydescribedinpreviouseditionsandtodescribethenewlyarisenones. Whenpreparingthisfourthedition,wehavemaintainedourinitialaimofrelyingonthe experience of a significant group of leading experts in the methodologies described, to provide a collection of step-by-step Arabidopsis protocols that can become an instructive and valuable laboratory companion. In this edition, the protocols have been grouped into fourdifferentclasses,thefirstonebeingdedicatedtoguidingtheaccesstoavailablepublic resources. The second class is devoted to genetic techniques and the third class covers different molecular and cell biological techniques. The last class of the book is dedicated tobiochemicalandphysiologicaltechniques.Asinpreviouseditions,wehaveincludedinall classes both methodologies that are widely used and those novel ones likely to open new avenuesofknowledgeandintenseworkinthenear future. We hope that the protocols presented in this new edition will continue to help alike experiencedresearchersandstudentsinthefieldofplantbiologybyclearlydescribingboth new and up-to-date techniques. Once more, we are extremely grateful to our colleagues who with their contributions have made this book possible, for their efforts, patience, and understanding. Madrid,Spain JoseJ.Sanchez-Serrano JulioSalinas v Contents Preface ..................................................................... v Contributors................................................................. ix PART I ARABIDOPSIS RESOURCES 1 HandlingArabidopsisandOtherBrassicaceae:Growth, PreservationofSeeds,Transformation,andGeneticCrosses.................. 3 ChristopherCalhoun,DeborahCrist,EmmaKnee, JulieMiller,EvaNagy,andDavidE.Somers 2 BioinformaticToolsinArabidopsisResearch................................ 25 G.AlexMason,AlexCant(cid:1)o-Pastor,SiobhanM.Brady, andNicholasJ.Provart PART II GENETIC TECHNIQUES IN ARABIDOPSIS 3 AnalysesofNaturalVariation:FieldExperiments andNucleotideDiversityforYourFavoriteGene ........................... 93 CarlosAlonso-Blanco,Bele´nMe´ndez-Vigo,andF.XavierPic(cid:1)o 4 Non-sterileGraftingMethodsforArabidopsis.............................. 113 Nien-ChenHuangandTien-ShinYu 5 CRISPR/Cas9-BasedGenomeEditingToolbox forArabidopsisthaliana ................................................. 121 DaisukeMiki,GauravZinta,WenxinZhang,FangnanPeng, ZhengyanFeng,andJian-KangZhu 6 TransientTransformationofA.thalianaSeedlings byVacuumInfiltration................................................... 147 Ce´sarBernat-Silvestre,VanessaDeSousaVieira,JuditSa´nchez-Simarro, FernandoAniento,andMarı´aJesu´sMarcote 7 TransientExpressionofFluorescentFusionProteins inArabidopsisProtoplasts................................................ 157 YonglunZeng,ChangyangJi,YoushunLin,andLiwenJiang 8 ArabidopsisPhotosyntheticandHeterotrophicCell SuspensionCultures..................................................... 167 EnricoCortese,LucaCarraretto,BarbaraBaldan, andLorellaNavazio 9 Genome-WideAssociationStudiesinArabidopsisthaliana: StatisticalAnalysisandNetwork-BasedAugmentationofSignals.............. 187 TakLeeandInsukLee vii viii Contents PART III MOLECULAR AND CELL BIOLOGICAL TECHNIQUES IN ARABIDOPSIS 10 VisualizingandMeasuringSingleLocusDynamics inArabidopsisthaliana .................................................. 213 AnisMeschichiandStefanieRosa 11 IdentificationandQuantificationofSmallRNAs............................ 225 DiSun,ZeyangMa,JiayingZhu,andXiurenZhang 12 FlowCytometryandSortinginArabidopsis................................ 255 DavidW.GalbraithandGuilingSun 13 LiveImagingofArabidopsisLeafandVegetativeMeristemDevelopment...... 295 MonicaPiaCaggiano,XiulianYu,CarolynOhno, PiaSappl,andMarcusG.Heisler 14 UsingGeneticallyEncodedFluorescentBiosensorsforQuantitative InVivoImaging........................................................ 303 AkiraYoshinari,JacobMoe-Lange,ThomasJ.Kleist, HeatherN.Cartwright,DavidA.Quint,DavidW.Ehrhardt, WolfB.Frommer,andMasayoshiNakamura 15 UsingTripartiteSplit-sfGFPfor theStudyofMembrane Protein–ProteinInteractions ............................................. 323 Tzu-YinLiu 16 High-PressureFreezingandFreezeSubstitutionforTransmission ElectronMicroscopyImagingandImmunogold-Labeling ................... 337 MarisaS.Otegui 17 AtomicForceMicroscopytoStudyCellWallMechanicsinPlants ............ 349 MateuszMajda 18 IdentificationandCharacterizationofReproductive MutationsinArabidopsis ................................................ 371 Marı´aFlores-Tornero,StefanieSprunck,andThomasDresselhaus PART IV BIOCHEMICAL AND PHYSIOLOGICAL TECHNIQUES IN ARABIDOPSIS 19 MetabolomicAnalysisofNaturalVariationinArabidopsis ................... 393 SiWu,SalehAlseekh,YarivBrotman,andAlisdairR.Fernie 20 UntargetedMetabolomicsofArabidopsisStomatalImmunity................ 413 LisaDavid,JianingKang,andSixueChen 21 Biotin-BasedProximityLabelingofProteinComplexesinPlanta............. 425 MadihaKhan,RajagopalSubramaniam,andDarrellDesveaux 22 PhosphoproteomicAnalysisofPlantMembranes ........................... 441 LinXi,WaltraudX.Schulze,andXuNaWu 23 RNA-BindingProteinImmunoprecipitationandHigh-Throughput Sequencing ............................................................ 453 TinoK¨osterandDorotheeStaiger Index ...................................................................... 463 Contributors CARLOSALONSO-BLANCO • DepartamentodeGene´ticaMoleculardePlantas,Centro NacionaldeBiotecnologı´a(CNB),ConsejoSuperiordeInvestigacionesCientı´ficas(CSIC), Madrid,Spain SALEHALSEEKH • Max-Planck-InstituteofMolecularPlantPhysiology,Potsdam-Golm, Germany;CenterofPlantSystemsBiologyandBiotechnology,Plovdiv,Bulgaria FERNANDOANIENTO • DepartamentodeBioquı´micayBiologı´aMolecular,Estructurade RecercaInterdisciplinarenBiotecnologı´aiBiomedicina(ERIBIOTECMED), UniversitatdeVale`ncia,Burjassot(Valencia),Spain BARBARABALDAN • DepartmentofBiology,UniversityofPadova,Padova,Italy;Botanical Garden,UniversityofPadova,Padova,Italy CE´SARBERNAT-SILVESTRE • DepartamentodeBioquı´micayBiologı´aMolecular,Estructurade RecercaInterdisciplinarenBiotecnologı´aiBiomedicina(ERIBIOTECMED), UniversitatdeVale`ncia,Burjassot(Valencia),Spain SIOBHANM.BRADY • DepartmentofPlantBiologyandGenomeCenter,Universityof California,Davis,Davis,CA,USA YARIVBROTMAN • DepartmentofLifeSciences,Ben-GurionUniversityoftheNegev, Beersheba,Israel MONICAPIACAGGIANO • EuropeanMolecularBiologyLaboratory,Heidelberg,Germany CHRISTOPHERCALHOUN • ArabidopsisBiologicalResourceCenter,Center forAppliedPlant Sciences,TheOhioStateUniversity,Columbus,OH,USA ALEXCANTO´-PASTOR • DepartmentofPlantBiologyandGenomeCenter,Universityof California,Davis,Davis,CA,USA LUCA CARRARETTO • DepartmentofBiology,UniversityofPadova,Padova,Italy HEATHERN.CARTWRIGHT • DepartmentofPlantBiology,CarnegieInstitutionforScience, Stanford,CA,USA SIXUECHEN • DepartmentofBiology,UniversityofFlorida,Gainesville,FL,USA; UniversityofFloridaGeneticsInstitute(UFGI),Gainesville,FL,USA;PlantMolecular andCellularBiologyProgram,UniversityofFlorida,Gainesville,FL,USA;Proteomics andMassSpectrometry,InterdisciplinaryCenter forBiotechnologyResearch(ICBR), UniversityofFlorida,Gainesville,FL,USA ENRICO CORTESE • DepartmentofBiology,UniversityofPadova,Padova,Italy DEBORAHCRIST • ArabidopsisBiologicalResourceCenter,CenterforAppliedPlantSciences, TheOhioStateUniversity,Columbus,OH,USA LISADAVID • DepartmentofBiology,UniversityofFlorida,Gainesville,FL,USA;University ofFloridaGeneticsInstitute(UFGI),Gainesville,FL,USA DARRELLDESVEAUX • DepartmentofCellandSystemsBiology,UniversityofToronto, Toronto,ON,Canada;Centrefor theAnalysisofGenomeEvolutionandFunction, UniversityofToronto,Toronto,ON,Canada THOMASDRESSELHAUS • CellBiologyandPlantBiochemistry,RegensburgCenter for Biochemistry,UniversityofRegensburg,Regensburg,Germany DAVIDW.EHRHARDT • DepartmentofBiology,StanfordUniversity,Stanford,CA,USA; DepartmentofPlantBiology,CarnegieInstitutionforScience,Stanford,CA,USA ix x Contributors ZHENGYANFENG • ShanghaiCenter forPlantStressBiology,CenterofExcellencefor MolecularPlantSciences,ChineseAcademyofSciences,Shanghai,China ALISDAIR R.FERNIE • Max-Planck-InstituteofMolecularPlantPhysiology,Potsdam-Golm, Germany;CenterofPlantSystemsBiologyandBiotechnology,Plovdiv,Bulgaria MARI´AFLORES-TORNERO • CellBiologyandPlantBiochemistry,RegensburgCenter for Biochemistry,UniversityofRegensburg,Regensburg,Germany WOLFB.FROMMER • InstituteofTransformativeBio-Molecules(WPI-ITbM),Nagoya University,Nagoya,Japan;InstituteforMolecularPhysiology,Heinrich-Heine- Universita€tDu¨sseldorf,Du¨sseldorf,Germany DAVIDW.GALBRAITH • UniversityofArizona,SchoolofPlantSciencesandBio5Institute, Tucson,AZ,USA;HenanUniversity,InstituteofPlantStressBiology,SchoolofLife Sciences,Kaifeng,China MARCUS G.HEISLER • SchoolofLifeandEnvironmentalSciences,UniversityofSydney, Sydney,NSW,Australia;EuropeanMolecularBiologyLaboratory,Heidelberg,Germany NIEN-CHEN HUANG • InstituteofPlantandMicrobialBiology,AcademiaSinica,Taipei, Taiwan LIWENJIANG • CentreforCellandDevelopmentalBiology,StateKeyLaboratoryof Agrobiotechnology,SchoolofLifeSciences,TheChineseUniversityofHongKong,Shatin, NT,HongKong CHANGYANGJI • CentreforCellandDevelopmentalBiology,StateKeyLaboratoryof Agrobiotechnology,SchoolofLifeSciences,TheChineseUniversityofHongKong,Shatin, NT,HongKong JIANINGKANG • UniversityofFloridaGeneticsInstitute(UFGI),Gainesville,FL,USA; CollegeofLifeScience,NortheastAgriculturalUniversity,Harbin,China MADIHAKHAN • OttawaResearchandDevelopmentCentre,AgricultureandAgri-Food Canada,Ottawa,ON,Canada;DepartmentofCellandSystemsBiology,Universityof Toronto,Toronto,ON,Canada THOMASJ.KLEIST • InstituteforMolecularPhysiology,Heinrich-Heine-Universita€t Du¨sseldorf,Du¨sseldorf,Germany EMMA KNEE • ArabidopsisBiologicalResourceCenter,Center forAppliedPlantSciences, TheOhioStateUniversity,Columbus,OH,USA TINO KO¨STER • FacultyofBiology,RNABiologyandMolecularPhysiology,Bielefeld University,Bielefeld,Germany INSUKLEE • DepartmentofBiotechnology,CollegeofLifeScienceandBiotechnology,Yonsei University,Seoul,Korea TAKLEE • DepartmentofBiotechnology,CollegeofLifeScienceandBiotechnology,Yonsei University,Seoul,Korea;SainsburyLaboratory,UniversityofCambridge,Cambridge,UK YOUSHUNLIN • CentreforCellandDevelopmentalBiology,StateKeyLaboratoryof Agrobiotechnology,SchoolofLifeSciences,TheChineseUniversityofHongKong,Shatin, NT,HongKong TZU-YINLIU • DepartmentofLifeScienceandInstituteofBioinformatics andStructural Biology,NationalTsingHuaUniversity,Hsinchu,Taiwan MATEUSZMAJDA • DepartmentofCellandDevelopmentalBiology,JohnInnesCentre, Norwich,UK MARI´AJESU´SMARCOTE • DepartamentodeBioquı´micayBiologı´aMolecular,Estructurade RecercaInterdisciplinarenBiotecnologı´aiBiomedicina(ERIBIOTECMED), UniversitatdeVale`ncia,Burjassot(Valencia),Spain