ARABIDOPSIS PROTOCOLS.

Preface Contents Contributors Part I: Arabidopsis Resources Chapter 1: Handling Arabidopsis and Other Brassicaceae: Growth, Preservation of Seeds, Transformation, and Genetic Crosses 1 Introduction 2 Materials 2.1 Sterilization, Cell Culture, Plant Growth, and Seed Harvest 2.2 Control of Environmental Growth Conditions for Optimal Plant Growth 2.3 Preparation of Seeds for Short- and Long-Term Storage 2.4 Seed Quality Control 3 Methods 3.1 Growth of Arabidopsis and Other Brassicaceae Plants and Cultures 3.1.1 Liquid and Gas Sterilization of Arabidopsis Seeds for Growth on Solid Media 3.1.2 Growth of Arabidopsis Cells in Culture 3.1.3 Planting Seeds of Arabidopsis and Other Members of the Brassicaceae on Soil 3.2 Growth Conditions 3.2.1 Maintenance of Plants in Growth Area 3.2.2 Monitoring Environmental Growth Conditions 3.3 Seed Handling 3.3.1 Plant Isolation, Harvesting, and Preparation for Storage Plant Isolation Harvesting Preparation for Storage 3.3.2 Seed Storage and Preservation 3.4 Seed Quality Control 3.4.1 Environmental Data Analysis 3.4.2 Microscopy 3.4.3 Germination Testing 3.5 Genetic Crosses 3.6 Floral Dip Transformation of Arabidopsis with Agrobacterium tumefaciens 4 Notes References Chapter 2: Bioinformatic Tools in Arabidopsis Research 1 Introduction 2 Materials 3 Methods 3.1 Genome Databases 3.1.1 Araport 3.1.2 1001 Genomes 3.2 Precomputed Gene Trees 3.2.1 Ensembl Plants 3.2.2 PLAZA 3.2.3 PANTHER 3.3 Epigenomic Tools 3.3.1 EPIC-CoGe Browser for Arabidopsis Epigenomic Data 3.4 Expression Analysis 3.4.1 eFP Browser 3.4.2 eFP-Seq Browser 3.4.3 TraVA 3.4.4 Genevestigator 3.5 Coexpression Tools 3.5.1 Expression Angler 3.5.2 ATTED II 3.5.3 AraNet 3.5.4 AtCAST2 3.6 Promoter Analysis 3.6.1 Cistome 3.6.2 MEME-Suite FIMO AME- Analysis of Motif Enrichment 3.6.3 ePlant Promoter Analysis 3.6.4 TAIR Motif Analysis 3.7 Functional Classification 3.7.1 AgriGO 3.7.2 AmiGO 3.7.3 Classification SuperViewer 3.8 Pathway Visualization 3.8.1 AraCyc 3.8.2 Pathway Visualization: MapMan 3.9 Protein Information 3.9.1 Protein Information: SUBA4 3.9.2 Protein Information: Cell eFP Browser 3.9.3 P3DB: Plant Protein Phosphorylation Database 3.9.4 Plant PTM Viewer 3.10 Protein-Protein Interaction Networks 3.10.1 Arabidopsis Interactions Viewer 2 (AIV2) 3.11 Integrated Tools 3.11.1 VirtualPlant 3.11.2 GeneMania 3.11.3 ePlant 3.11.4 TF2Network 3.12 Targeting Tools for Confirming Gene Function 3.12.1 CRISPR 3.12.2 Artificial miRNA 3.12.3 SIGnAL T-DNA Express 3.13 Conclusions 4 Notes References Part II: Genetic Techniques in Arabidopsis Chapter 3: Analyses of Natural Variation: Field Experiments and Nucleotide Diversity for Your Favorite Gene 1 Introduction 2 Materials 3 Methods 3.1 Growth of Arabidopsis in Field Experiments for Phenotypic Analyses 3.2 Analyses of Nucleotide Diversity and Environmental Associations for Your Favorite Genes 4 Notes References Chapter 4: Non-sterile Grafting Methods for Arabidopsis 1 Introduction 2 Materials 3 Methods 3.1 Pin-Fasten Epicotyl Grafting 3.2 Pin-Fasten Hypocotyl Grafting 4 Notes References Chapter 5: CRISPR/Cas9-Based Genome Editing Toolbox for Arabidopsis thaliana 1 Introduction 2 Materials 2.1 Vector Construction for Single/Multiple Gene Mutations and Base Editing 2.2 Agrobacterium Transformation 2.3 Arabidopsis Transformation 2.4 Screening of Mutations or Base Editings 2.5 Generation of Gene Targeting Constructs 3 Methods 3.1 Single/Multiple Gene Mutations and Base Editing 3.1.1 Design of sgRNA 3.1.2 sgRNA Order and Dilution 3.1.3 Preparation of sgRNA Construct 3.1.4 Preparation of T-DNA Construct for Multiple Targets (Figs. 2 and 7) 3.1.5 Agrobacterium-Mediated Arabidopsis Transformation 3.1.6 Screening of Mutations or Base Editings 3.2 Gene Targeting 3.2.1 Design of sgRNA 3.2.2 Design of Donor DNA 3.2.3 Preparation of Donor T-DNA Construct for Agrobacterium Transformation 3.2.4 Screening of Gene-Targeted Arabidopsis Plants 4 Notes References Chapter 6: Transient Transformation of A. thaliana Seedlings by Vacuum Infiltration 1 Introduction 2 Materials 2.1 Buffers, Solutions and Media 2.2 Equipment 2.3 Plants, Agrobacterium Strains, and Vectors 3 Methods 3.1 Growth of Seedlings Before Infiltration 3.2 Preparation of Agrobacterium Cultures for Agroinfiltration 3.2.1 Agrobacterium Culture Growth 3.2.2 Agrobacterium Suspension for Agroinfiltration 3.3 Agroinfiltration 3.4 Image Acquisition 4 Notes References Chapter 7: Transient Expression of Fluorescent Fusion Proteins in Arabidopsis Protoplasts 1 Introduction 2 Materials 2.1 Arabidopsis PSBD Cell Culture 2.2 Protoplast Isolation 2.3 Protoplast Transfection 2.4 Super-Resolution Confocal Microscopy Imaging 3 Methods 3.1 Protoplast Isolation 3.2 Protoplast Transfection 3.3 Confocal Imaging 3.4 Super-Resolution Confocal Microscopy Imaging 4 Notes References Chapter 8: Arabidopsis Photosynthetic and Heterotrophic Cell Suspension Cultures 1 Introduction 2 Materials 2.1 Growth Facilities 2.2 Preparation of Phytohormones 2.3 Preparation of Culture Media for Photosynthetic Cultures 2.4 Preparation of Culture Media for Heterotrophic Cultures 2.5 Preparation of Plates Containing Agarized Medium for Photosynthetic Cultures 2.6 Preparation of Plates Containing Agarized Medium for Heterotrophic Cultures 2.7 Surface Sterilization of Seeds and Sowing on Petri Dishes 2.8 Preparation of Enzymatic Solution and Protoplast Resuspension Buffer 3 Methods 3.1 Sterility Conditions 3.2 Establishment and Maintenance of Photosynthetic Cell Suspension Cultures 3.2.1 Surface Sterilization of Seeds 3.2.2 Sowing on Petri Dishes and Induction of Green Calli 3.2.3 Initiation of Photosynthetic Cell Suspension Cultures 3.2.4 Setup and Subculturing of Photosynthetic Cell Suspension Cultures 3.2.5 Determination of the Cell Suspension Culture Growth Curve 3.3 Establishment and Maintenance of Heterotrophic Cell Suspension Cultures 3.3.1 Surface Sterilization of Seeds, Sowing on Petri Dishes, and Growing of Seedlings 3.3.2 Induction and Maintenance of Heterotrophic Calli 3.3.3 Initiation and Subculturing of Heterotrophic Cell Suspension Cultures 3.4 Interconversion of Photosynthetic and Heterotrophic Cell Suspension Cultures 3.4.1 Pulse Amplitude Modulation (PAM) Fluorometry 3.5 Isolation of Protoplasts from Cell Suspension Cultures 4 Notes References Chapter 9: Genome-Wide Association Studies in Arabidopsis thaliana: Statistical Analysis and Network-Based Augmentation of Sig... 1 Introduction 2 Materials 2.1 Arabidopsis Ecotype Accessions 2.2 Basic Computational Requirements 2.3 Software for GWAS Analysis 3 Methods 3.1 Obtaining and Preprocessing Genotype Data 3.1.1 Download Genotype Data for Arabidopsis Accessions 3.1.2 Download PLINK Software 3.1.3 Remove Short Indels and Retain SNPs Only 3.1.4 Filter Out SNPs That Have Minor Allele Frequency of Lower Than 10% 3.1.5 Modify .map File 3.1.6 Generate .tfam and .tped File for Further Analysis 3.2 Selecting Natural Accessions for GWAS 3.3 Evaluation of Population Structure 3.3.1 Calculate Eigenvectors and Eigenvalues with PLINK 3.3.2 Draw PCA Plot 3.4 Obtaining Phenotype Data 3.5 Running GWAS 3.5.1 EMMAX Installation 3.5.2 Create a Kinship Matrix 3.5.3 Input Phenotype Data 3.5.4 Run EMMAX 3.6 Evaluation of GWAS Results with Quantile-Quantile Plot (Q-Q Plot). 3.6.1 Run R and Install qqman with the Following Command 3.6.2 Run R and the Plotting Process 3.7 Evaluation of GWAS Results with a Manhattan Plot 3.8 Selecting Significant SNPs with the Multiple Hypothesis Adjustment 3.8.1 Bonferroni Correction 3.8.2 Sidk Correction 3.8.3 Benjamini-Hochberg Correction 3.9 Assigning SNPs to Genes 3.9.1 Obtain Genomic Positions of Arabidopsis Genes from TAIR 3.9.2 Select the Lines That Have Information on the Whole Gene Position 3.9.3 Write a Script 3.10 Augmenting GWAS Signals with araGWAB 3.10.1 GWAS Boosting using the araGWAB Web Application 3.10.2 Interpreting and Downloading Results 4 Notes References Part III: Molecular and Cell Biological Techniques in Arabidopsis Chapter 10: Visualizing and Measuring Single Locus Dynamics in Arabidopsis thaliana 1 Introduction 2 Materials 2.1 Plant Growth 2.2 Microscopy 2.3 Data Analysis 3 Methods 3.1 Plant Growth and Sample Preparation 3.2 Image Acquisition 3.3 Image Processing 3.4 Data Analysis 4 Notes References Chapter 11: Identification and Quantification of Small RNAs 1 Introduction 2 Materials 2.1 Synthesis of the 5′ Pre-adenylated 3′ Adapter Oligonucleotides 2.1.1 Synthesis of the Adenosine-5′-phosphoimidazolide (ImpA) 2.1.2 Quality Control of ImpA 2.1.3 Adenylation of 3′ Adapter Oligonucleotides 2.2 Primer Purification 2.3 Marker Labeling 2.3.1 CIP Treatment of RNA Oligomers with 5′ Mono- or Tri-phosphate 2.3.2 T4 PNK Treatment 2.4 RNA Extraction 2.5 Preparation of RNA with Radiolabeled Marker Cocktail and Size Selection 2.6 3′ Ligation 2.7 5′ Ligation 2.8 Reverse Transcription 2.9 PCR 2.10 Pme I Digestion 2.11 Computing Analysis Workflow 3 Methods 3.1 Synthesis of the 5′ Pre-adenylated 3′ Adapter Oligonucleotides 3.1.1 Synthesis of the Adenosine-5′-Phosphoimidazolide (ImpA) 3.1.2 Quality Control of ImpA 3.1.3 Adenylation of 3′ Adapter Oligonucleotides (See Note 1) 3.2 Primer Purification 3.3 Marker Labeling 3.3.1 CIP Treatment of RNA Oligomers with 5′ Mono- or Tri-phosphate (See Note 6) 3.3.2 T4 PNK Treatment 3.4 RNA Extraction 3.5 Preparation of RNA with Radiolabeled Marker Cocktail and Size Selection 3.6 3′ Ligation 3.7 5′ Ligation 3.8 Reverse Transcription 3.9 PCR 3.10 Pme I Digestion 3.11 Computing Analysis Workflow 4 Notes References Chapter 12: Flow Cytometry and Sorting in Arabidopsis 1 Introduction 1.1 Applying Flow Cytometry and Cell Sorting to Vascular Plants 1.2 Flow Cytometry and Sorting Applied to Plant Protoplasts 1.3 Analyzing Protoplasts Using Flow Cytometry 1.4 Identifying Specific Cell Types in Flow Cytometry and Sorting 1.5 Dealing with Large Cells 1.6 Dealing with Superlarge Objects, such as Seed 1.7 Flow Cytometry and Sorting of Plant Homogenates 1.8 Dealing with Low Amounts of RNA for Expression Profiling 1.9 Special Cases: Flow Analysis and Sorting of Pollen and Sperm Cells 1.10 Major Equipment 2 Materials 2.1 Preparation of Protoplasts 2.2 Isolation of Nuclei 2.3 Preparing Nuclei for 10x Genomics Processing 2.4 Extraction and Amplification of RNA from Protoplasts and Nuclei 2.5 Flow Cytometry Standards 3 Methods 3.1 Preparation of Protoplasts (See Notes 2 and 3) 3.2 Preparation of Plant Homogenates and Flow Analysis of Nuclei 3.2.1 Instrument Settings for Flow Cytometric Analyses of Nuclei Using the Accuri C6 Using the CytoFLEX 3.2.2 Instrument Settings for Spectral Analysis of Nuclei using the SA3800 3.3 Flow Sorting of Protoplasts, Pollen and Nuclei 3.3.1 Analysis and Sorting of Arabidopsis Protoplasts and Pollen using the S3 3.3.2 Analysis and Sorting of Nuclei using the S3 3.3.3 Processing Sorted Protoplasts and Nuclei 3.4 Flow Analysis of Protoplasts and Nuclei Expressing Fluorescent Proteins other than GFP 3.5 Amplification of Targets from Sorted Protoplasts and Nuclei 3.5.1 Preparation of RNA Targets from Populations of Sorted Protoplasts and Nuclei 3.5.2 Preparation of RNA Targets from Single Sorted Protoplasts and Nuclei 3.6 Analysis of Gene Expression in Populations of Single Nuclei using the Chromium System 4 Notes References Chapter 13: Live Imaging of Arabidopsis Leaf and Vegetative Meristem Development 1 Introduction 2 Materials 3 Methods 3.1 Seed Germination 3.2 Seedling Preparation 3.3 Time-Lapse Imaging of Leaves and Vegetative Meristems 4 Notes References Chapter 14: Using Genetically Encoded Fluorescent Biosensors for Quantitative In Vivo Imaging 1 Introduction 1.1 Types of Genetically Encoded Fluorescent Biosensors 1.1.1 Fluorescent Proteins 1.1.2 Indirect Fluorescent Biosensors 1.1.3 FRET-Based Biosensors 1.1.4 Intrinsic Biosensors 1.1.5 Extrinsic Intensiometric Biosensors 1.1.6 Improvement of Intensiometric Biosensors 1.1.7 Activity Sensors 1.2 How to Use Genetically Encoded Fluorescent Biosensors 1.2.1 Selection of Genetic Background 1.2.2 Selection of Promoter 1.2.3 The Use of Affinity Variants of the Sensors 1.2.4 Potential of Biosensors to Act as Buffers or as Components of Signaling Networks 2 Materials 2.1 Perfusion Chamber System 2.1.1 Perfusion Chamber for a Simplified Version of the RootChip 2.1.2 Perfusion Chamber Holder 2.1.3 Perfusion System 2.2 Microscope Equipment 2.3 Plant Material, Growth Medium, and Perfusion Solutions 3 Methods 3.1 Preparation of Plastic Cones 3.2 Sowing and Germination 3.3 Transfer of Seedlings onto Perfusion Chamber 3.4 Mount the Perfusion Chamber onto the Microscope 3.4.1 Preparation of Perfusion Chamber 3.4.2 Assemble the Perfusion Chamber and Chamber Holder 3.4.3 Create a Constant Flow of the Liquid Medium 3.5 Microscopy 3.6 Image Analysis 3.6.1 Background Subtraction 3.6.2 Make Graph 3.6.3 Normalization by LSSmOrange Intensity and Color Adjustment 3.6.4 Make Kymograph 4 Notes References Chapter 15: Using Tripartite Split-sfGFP for the Study of Membrane Protein-Protein Interactions 1 Introduction 2 Materials 2.1 Binary Vectors and Constructs 2.2 Agrobacterium Culture and Infiltration 2.3 Plant Culture 2.4 Consumables 2.5 Equipment 2.6 Antibiotics and Reagent Stock Solution 3 Methods 3.1 Transformation and Culture of Agrobacterium 3.2 Growing of Nicotiana Benthamiana 3.3 Infiltration of Nicotiana Benthamiana 3.4 Microscopic Imaging and Tripartite Split-GFP Assay 4 Notes References Chapter 16: High-Pressure Freezing and Freeze Substitution for Transmission Electron Microscopy Imaging and Immunogold-Labeling 1 Introduction 2 Materials 2.1 Plant-Related Materials 2.2 High-Pressure Freezing 2.3 Freeze-Substitution and Resin Embedding 2.4 Preparation of Sections for Electron Microscopy 2.5 Immunolabeling 3 Methods 3.1 Plant Material 3.1.1 Arabidopsis Seedlings: Root Tips and Cotyledons 3.1.2 Developing Seeds 3.1.3 Developing Anthers 3.2 High-Pressure Freezing 3.3 Freeze-Substitution and Resin Embedding 3.3.1 For Structural Analysis Using Dry Ice and Styrofoam Box 3.3.2 For Immunolabeling Using the Leica AFS 3.4 Post-Embedding Sectioning and Heavy Metal Staining for Structural Analysis 3.5 Immunolabeling 4 Notes References Chapter 17: Atomic Force Microscopy to Study Cell Wall Mechanics in Plants 1 Introduction 2 Materials 2.1 Plant Cultivation 2.2 Sample Preparation 2.3 AFM Hardware and Cantilevers 2.4 Software 3 Methods 3.1 Plant Material Cultivation and Fixation 3.2 Embedding Leaf Pieces in Resin and Sectioning 3.3 Selecting the Cantilever and Its Calibration 3.4 Acquiring Images and Force Curves 3.5 Extracting Apparent Elastic Modulus from Force Curves 4 Notes References Chapter 18: Identification and Characterization of Reproductive Mutations in Arabidopsis 1 Introduction 2 Materials 2.1 Silique Dissection 2.2 Male Gametophyte Development and Viability 2.3 In Vivo and In Vitro Pollen Tube Growth 2.4 Female Gametophyte and Embryo Development 3 Methods 3.1 Silique Dissection and Observation 3.1.1 Dissection of Siliques for Microscopy 3.1.2 Fixation and Clearing of Siliques for Microscopy 3.2 Male Gametophyte Development 3.2.1 Pollen Morphology and Content 3.2.2 FDA Staining for Pollen Viability 3.2.3 Alexander Staining for Pollen Viability 3.2.4 In Vitro Pollen Germination 3.2.5 In Vivo Pollen Tube Growth 3.3 Ovule and Embryo Sac Development 3.4 Embryo Development 4 Notes References Part IV: Biochemical and Physiological Techniques in Arabidopsis Chapter 19: Metabolomic Analysis of Natural Variation in Arabidopsis 1 Introduction 2 Materials 2.1 Plant material 2.2 GC-MS 2.2.1 Sampling and Extraction 2.2.2 Derivatization 2.2.3 GC-TOF-MS 2.3 LC-MS 2.3.1 Sampling and Extraction 2.3.2 LC-MS Analysis 2.3.3 Data Analysis 3 Methods 3.1 Experimental Design 3.2 Plant Material and Sampling 3.3 Sample Processing and Extraction 3.4 Sample Preparation and Analysis 3.5 Data Processing 3.6 Data Normalization 3.7 GWAS Mapping 4 Notes References Chapter 20: Untargeted Metabolomics of Arabidopsis Stomatal Immunity 1 Introduction 2 Materials 2.1 Bacterial Treatment of Plants 2.2 Harvesting of Guard Cell Samples 2.3 Metabolite Extraction 2.4 Mass Spectrometry Using Acquire X 2.5 Data Analysis 3 Methods 3.1 Bacterial Treatment of Plants (See Note 1) 3.2 Harvesting of Guard Cell Samples (See Note 2) 3.3 Metabolite Extraction (See Note 3) 3.4 Mass Spectrometry Using Acquire X (See Note 4) 3.5 Data Analysis (See Note 5) 4 Notes References Chapter 21: Biotin-Based Proximity Labeling of Protein Complexes in Planta 1 Introduction 2 Materials 2.1 Inducible Vector Cloning 2.2 Plant Transformation 2.3 Transgenic Arabidopsis Selection and Expression 2.4 Transgenic Arabidopsis Plants for Protein Interaction Tests 2.5 Biotin Infiltration of Arabidopsis Plants 2.6 Dexamethasone Induction of pDB Constructs 2.7 Protein Extraction 2.8 Removing Excess Biotin from Protein Extract 2.9 Affinity Purification 2.10 Trypsin Digest for Mass Spectrometry 3 Methods 3.1 BioID Vector Cloning 3.2 Plant Transformation 3.3 Transgenic Arabidopsis Selection and Expression 3.4 Biotin Infiltration 3.5 DEX Induction 3.6 Protein Extractions 3.7 Removing Excess Biotin from Protein Extract 3.8 Affinity Purification with Streptavidin-Sepharose Beads 3.9 Trypsin Digestion for Mass Spectrometry 4 Notes References Chapter 22: Phosphoproteomic Analysis of Plant Membranes 1 Introduction 2 Materials 2.1 Buffers and Solutions 2.2 Other Materials 2.3 Specific Equipment 3 Methods 3.1 Preparation of Microsomal Fraction (Timing ~2-3 h) 3.2 In Solution Trypsin Digestion (Timing ~17 h) 3.3 TiO2 Phosphopeptide Enrichment (Timing ~2 h) 3.4 Peptide Desalting Using C18 StageTips (Timing ~1.5 h) 3.5 LC-MS/MS Analysis and Peptide Identification (Timing ~ 2.5 h Per Sample) 3.6 Label-Free Relative Quantification Analysis 4 Notes References Chapter 23: RNA-Binding Protein Immunoprecipitation and High-Throughput Sequencing 1 Introduction 2 Materials 2.1 Plant Growth 2.2 Fixation 2.3 Preparation of Beads 2.4 Total Extract Preparation 2.5 Immunoprecipitation 2.6 RNA Isolation 2.7 Library Preparation 3 Methods 3.1 Plant Growth 3.2 Fixation 3.3 Preparation of Beads 3.4 Total Extract Preparation 3.5 Immunoprecipitation 3.6 RNA Isolation 3.7 Libraries 4 Notes References Index "This fourth edition compiles the most recent methodologies developed to exploit the Arabidopsis genome. Chapters detail access to public resources, to genetic, cell biology, biochemical and physiological techniques, and sections on genome, transcriptome, proteome, metabolome and other whole-system approaches. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Arabidopsis Protocols, Fourth Edition aims to ensure successful results in the further study of this vital field."--Back cover