Preface
Contents
Contributors
Part I: Review and Overview Chapters
Chapter 1: Flower Development in Arabidopsis
1 Part 1: The Floral Transition
1.1 External Parameters Influencing Flowering-Temperature and Photoperiod
1.1.1 Temperature
1.1.2 Photoperiod
1.2 Internal Cues-Metabolism and Age-Dependent Pathways
1.2.1 Sugar and Nitrate Metabolism
1.2.2 Age-Dependent Pathway
1.3 Integration of the Different Input to Promote Flowering and Flower Formation
2 Part 2: The First Hours of Arabidopsis Flowers
2.1 Primordium Positioning and Auxin Signaling
2.2 Auxin Biosynthesis and Transport
2.3 Downstream of Auxin Signaling
2.4 Boundaries and Polarization Set the Limit of the Floral Primordium
2.5 Establishment of a Transient Stem Cell Identity
2.6 Modifications in Cell Wall Composition Trigger Floral Outgrowth
3 Part 3: Determination of the Floral Primordia Identity
3.1 Activation of the Floral Meristem Identity
3.2 Repression of the Inflorescence Trait
3.3 A Common Early Regulatory Network Between LFY and AP1
3.4 The Genesis of the Boundary FM-SAM
4 Part 4: Establishment of the Floral Patterning-from the Historical ABC to the Revised ABCDE Model
4.1 Sepal Identity and Maintenance
4.2 Sepal Growth
4.3 Petal Identity and Maintenance
4.4 Petal Growth
4.5 Stamen Identity and Maintenance
4.6 Anther and Stamen Development
4.7 Nectary Formation
4.8 Carpel and Ovule Identity and Maintenance
4.9 MADS TF Target Gene Specificity
4.10 Boundaries and Polarity Genes Are Key to Define Proper Organ Identity Domains
5 Part 5: Floral Meristem Termination
6 Conclusions and Perspectives
6.1 Coordination and Timing Are Key
6.2 Rethinking MADS-Box TF Specificity
6.3 Reaching the Single Cell Level
6.4 The Cost of Making a Flower
References
Chapter 2: Flower Development in the Solanaceae
1 The Solanaceae Family (Nightshades): Crops and Model Species
2 Specification of Floral Organ Identity: Revisions to the ABC Model
2.1 Redefining the A-Function
2.2 Variations on the B-Function: Specializing for a Single Floral Organ
2.3 Multiple Functions for C-Class Genes
2.4 Uncovering the E-Function
2.5 Divergence to the Textbook ABC Model
3 Initiation and Fusion of Floral Organs
4 Growth and Maturation of Floral Organs: Chinese Lanterns and Petal Colors
5 Conclusion
References
Chapter 3: The ABC of Flower Development in Monocots: The Model of Rice Spikelet
1 Introduction
2 The Spikelet and the Flower in Grasses
3 The Spikelet and the Flower in Rice
4 Genetic Control of Spikelet and Floral Organ Identity in Rice: An ABC View
4.1 C and D Function Genes of Rice
4.2 B Function Genes of Rice
4.3 Rice SEP, AP1/SQUA, AGL6, and OsMADS32 Genes Specify Together the (A) Function
References
Chapter 4: Model Species to Investigate the Origin of Flowers
1 Introduction
1.1 What Is a Flower?
1.2 The Rise of Flowering Plants: When, Where, and Why?
1.3 A Family Tree of the Flowering Plants
1.4 A Portrait of the Ancestral Flower
2 Models and Molecular Approaches to Study the Origin of Flowers
2.1 Amborellales
2.2 Nymphaeales
2.3 Austrobaileyales
2.4 Magnoliids
2.5 Gymnosperms
3 Missing Links and New Approaches
References
Chapter 5: Hormones and Flower Development in Arabidopsis
1 Introduction
2 Floral Meristem Initiation
3 Sepals
4 Petals
5 Stamens
6 Gynoecium
7 Conclusions
References
Part II: Genetic and Phenotypic Analyses
Chapter 6: Genetic Screens for Floral Mutants in Arabidopsis thaliana: Enhancers and Suppressors
1 Introduction
1.1 Mutagenesis of Arabidopsis
1.1.1 EMS Mutagenesis of Arabidopsis
1.1.2 T-DNA Insertional Mutagenesis
1.2 Identification of the Mutation Responsible for the Observed Mutant Phenotype
1.2.1 Map-Based Positional Cloning
1.2.2 Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR)
1.2.3 Mapping by Deep Sequencing
2 Materials
2.1 Mutagenesis, Mutant Screening, and Initial Mapping of Mutations
2.1.1 EMS Mutagenesis of Arabidopsis
2.1.2 Planting EMS Mutagenized M1, M2, and Mapping Population Seeds
2.1.3 Preparing DNA for Map-Based Positional Cloning
2.1.4 CTAB DNA Extraction
2.1.5 Quick and Dirty PCR
2.1.6 T-DNA Insertional Mutagenesis: Agrobacterium-Mediated Transformation
2.2 Pinpointing the Mutation That Causes the Phenotype
2.2.1 Map-Based Positional Cloning: PCR
2.2.2 TAIL-PCR
2.2.3 Mapping by Deep Sequencing
3 Methods
3.1 Mutagenesis, Mutant Screening, and Initial Mapping of Mutations
3.1.1 EMS Mutagenesis of Arabidopsis
3.1.2 Planting EMS Mutagenized M1, M2, and Mapping Population Seeds
3.1.3 Preparing DNA for Map-Based Positional Cloning
3.1.4 CTAB DNA Extraction
3.1.5 Quick and Dirty Extraction
3.1.6 T-DNA Insertional Mutagenesis: Transformation and Selection of Agrobacterium
3.1.7 T-DNA Insertional Mutagenesis: Preparation of Agrobacterium
3.1.8 T-DNA Insertional Mutagenesis: Floral Dip
3.1.9 T-DNA Insertional Mutagenesis- Selection of Transformants
3.2 Pinpointing the Mutation That Causes the Phenotype
3.2.1 Map-Based Positional Cloning-PCR
3.2.2 Map-Based Positional Cloning-Analysis
3.2.3 mhiTAIL-PCR
3.2.4 Mapping by Deep Sequencing: Library Preparation
4 Notes
References
Chapter 7: Genetic and Phenotypic Analysis of Shoot Apical and Floral Meristem Development
1 Introduction
2 Materials
2.1 Confocal Laser Scanning Microscopy of the Embryonic Meristem
2.2 Histological Sectioning of the Vegetative Meristem
2.3 Meristem Size Measurement
2.4 Vegetative Meristem RNA In Situ Hybridization
2.5 Confocal Laser Scanning Microscopy of the Inflorescence Meristem
2.6 Live Imaging Confocal Laser Scanning Microscopy of the Inflorescence Meristem
2.7 Scanning Electron Microscopy of the Inflorescence Meristem
2.8 Floral Organ Number Counting
3 Methods
3.1 Confocal Laser Scanning Microscopy of the Embryonic Meristem
3.1.1 Embryo Dissection
3.1.2 Tissue Staining and Rinsing
3.1.3 Tissue Dehydration and Clearing
3.1.4 Mounting and Imaging
3.2 Histological Sectioning of the Vegetative Meristem
3.2.1 Tissue Dissection and Fixation
3.2.2 Tissue Dehydration and Infiltration
3.2.3 Tissue Staining and Embedding
3.2.4 Tissue Sectioning
3.2.5 Toluidine Blue Staining
3.2.6 Mounting and Visualization
3.3 Meristem Size Measurement
3.4 Vegetative Meristem RNA In Situ Hybridization
3.4.1 Fixation of Tissue Sections
3.4.2 Tissue Dehydration
3.4.3 Tissue Embedding in Paraffin
3.4.4 Tissue Sectioning and Mounting
3.4.5 Riboprobe Preparation (See Note 40)
3.4.6 Riboprobe Synthesis
3.4.7 Slide Pre-Hybridization and Hybridization Treatments
3.4.8 Post-Hybridization Washes
3.4.9 Detection
3.5 Confocal Laser Scanning Microscopy of the Inflorescence Meristem
3.5.1 Tissue Fixation
3.5.2 Tissue Staining and Rinsing
3.5.3 Tissue Dehydration and Clearing
3.5.4 Mounting and Imaging
3.6 Live Imaging Confocal Laser Scanning Microscopy of the Inflorescence Meristem
3.6.1 Tissue Harvesting
3.6.2 Tissue Staining and Mounting
3.7 Scanning Electron Microscopy of the Inflorescence Meristem
3.7.1 Tissue Fixation
3.7.2 Tissue Rinsing and Dehydration
3.7.3 Critical Point Drying
3.7.4 Tissue Mounting
3.8 Floral Organ Number Counting
3.8.1 Flower Dissection and Counting
3.8.2 Data Analysis
4 Notes
References
Chapter 8: Cell Biological Analyses of Anther Morphogenesis and Pollen Viability in Arabidopsis and Rice
1 Introduction
2 Materials
2.1 Alexander Red Staining
2.2 Iodine Pollen Starch Detection
2.3 FDA Staining and Imaging
2.4 Scanning Electron Microscopy for Anther Structure, Anther Dehiscence, and Pollen Wall Structure
2.5 Examination of Anther Anatomy Using Semi-Thin Sections
2.6 Callose Staining
2.7 Staining of the Pollen Exine and Intine
2.8 Detection of Programmed Cell Death Using TUNEL Assay
2.9 Ultrathin Section and Transmission Electron Microscopy (TEM) for Observation of Tapetum and Pollen Morphology
2.10 Sample Preparation for Laser Capture Microdissection
3 Methods
3.1 Alexander Staining and Photography
3.2 Iodine Pollen Starch Detection
3.3 FDA Staining and Imaging
3.4 Scanning Electron Microscopy for Anther Structure, Anther Dehiscence, and Pollen Wall Structure
3.5 Anther Anatomy Using Semi-Thin Sections
3.6 Callose Staining
3.7 Staining of the Pollen Exine and Intine
3.8 Detection of Programmed Cell Death Using TUNEL Assay
3.9 Ultrathin Section and Transmission Electron Microscopy (TEM) for Observation of Tapetum and Pollen Morphology
3.10 Sample Preparation for Laser Capture Microdissection
4 Notes
References
Chapter 9: Isolation of Meiocytes and Cytological Analyses of Male Meiotic Chromosomes in Soybean, Lettuce, and Maize
1 Introduction
2 Materials
2.1 Plants
2.2 Isolation of Male Meiocytes from Soybean and Lettuce
2.3 Chromosome Spread and Immunostaining in Arabidopsis, Soybean, and Lettuce
2.4 Light Microscopy of Maize Meiosis
2.5 Fluorescent In Situ Hybridization (FISH) of Maize Chromosomes
2.5.1 Maize Meiotic Chromosome Spreading
2.5.2 Labeled Probes
2.5.3 Pretreatment of Chromosome Spreads
2.5.4 Hybridization
2.5.5 Stringent Wash and Mounting
3 Methods
3.1 Isolation of Meiocytes from Soybean and Lettuce
3.2 Chromosome Spread for Meiotic Chromosomes of Soybean and Lettuce
3.3 Immunostaining of Meiotic Proteins in Lettuce
3.4 Immunolocalization of DNA Methylation and Histone Modifications in Arabidopsis Meiocytes
3.5 Isolation and Observation of Maize Meiosis by Light Microscopy
3.6 Fluorescent In Situ Hybridization (FISH) for Maize
3.6.1 Maize Meiotic Chromosome Spreading
3.6.2 Pretreatment of Chromosome Spreads
3.6.3 Hybridization
3.6.4 Stringent Wash and Mounting
4 Notes
References
Chapter 10: Genetic and Phenotypic Analyses of Carpel Development in Arabidopsis
1 Introduction
2 Materials
2.1 Aniline Blue Staining of Arabidopsis Pollen Tubes
2.2 Cleared Tissue for Observation of Vascular Development
2.3 NPA Treatment
2.4 Lignin Staining
2.5 Genetic Analyses
3 Methods
3.1 Aniline Blue Staining of Arabidopsis Pollen Tubes
3.1.1 Material Collection
3.1.2 Tissue Fixation
3.1.3 Pistil Softening
3.1.4 Pistil Staining
3.1.5 Pistil Mounting and Visualization
3.2 Cleared Tissue for Observation of Vascular Development
3.2.1 Material Collection
3.2.2 Tissue Fixation
3.2.3 Tissue Clearing
3.2.4 Pistil Mounting and Visualization
3.3 NPA Treatment
3.3.1 Plant Growth and Preparation
3.3.2 Plant Treatment
3.3.3 Phenotype Visualization
3.4 Lignin Staining: Whole Mount Phloroglucinol Staining (Wiesner Stain)
3.4.1 Tissue Fixation
3.4.2 Fruit Staining
3.4.3 Fruit Lignin Visualization
3.5 Lignin Staining: Tissue Section
3.5.1 Tissue Fixation
3.5.2 Tissue Dehydration and Paraplast Embedding
3.5.3 Tissue Sectioning
3.5.4 Tissue Staining
3.6 Genetic Analyses
4 Notes
References
Chapter 11: Genetic and Phenotypic Analysis of Ovule Development in Arabidopsis
1 Introduction
2 Materials
2.1 Clearing of Ovules for Wholemount Analysis
2.2 Staining of Ovules for Confocal Analysis
2.2.1 Aniline Blue Staining for Callose
2.2.2 Renaissance Staining for Analysis of Cell Morphology
2.3 Embedding and Sectioning Ovule Tissues for Immunolabelling
2.4 Crosses to Marker Lines for Assessment of Ovule Cell Identity
2.5 Laser Dissection of Ovule Tissues for Transcriptomic Analysis
3 Methods
3.1 Clearing of Ovules for Wholemount Analysis
3.2 Staining of Ovules for Confocal Analysis
3.2.1 Aniline Blue Staining for Callose
3.2.2 Renaissance Staining for Analysis of Cell Morphology
3.3 Embedding and Sectioning Ovule Tissues for Immunolabelling
3.4 Crosses to Marker Lines for Assessment of Ovule Cell Identity
3.5 Laser Dissection of Ovule Tissues for Transcriptomic Analysis
4 Notes
References
Part III: Experimental Systems
Chapter 12: Floral Induction Systems for the Study of Arabidopsis Flower Development
1 Introduction
2 Materials
2.1 Plant Lines and Growth
2.2 Reagents for Induction of Flower Formation
2.3 Reagents for Agrobacterium-Mediated Transformation Using the Floral Dip Method
3 Methods
3.1 Plant Growth and Treatment
3.2 Agrobacterium-Mediated Transformation Using the Floral Dip Method
4 Notes
References
Chapter 13: Protoplasting and Fluorescence-Activated Cell Sorting of the Shoot Apical Meristem Cell Types
1 Introduction
2 Materials
2.1 Protoplasting and Cell Sorting
2.2 Isolation of Total RNA from Sorted Cells
3 Methods
3.1 Protoplasting and Cell Sorting
3.2 Isolation of Total RNA from Sorted Cells
4 Notes
References
Chapter 14: Protoplast Isolation for Plant Single-Cell RNA-seq
1 Introduction
2 Materials
2.1 Plant Growth and Tissue Collection
2.2 Protoplast Isolation
3 Methods
4 Notes
References
Chapter 15: Plant Nuclei Isolation for Single-Nucleus RNA Sequencing
1 Introduction
2 Materials
3 Method
4 Notes
References
Chapter 16: Isolation of Nuclei Tagged in Specific Cell Types (INTACT) in Arabidopsis
1 Introduction
2 Materials
2.1 Generation of INTACT Reporter Lines
2.2 Verification of INTACT Reporter Lines by Microscopy and In Situ Histochemistry
2.3 INTACT
2.4 Assessment of the Enrichment of SAM-Specific Nuclei by Low Input Smart-qPCR
3 Methods
3.1 Establishment of INTACT Reporter Plant Lines
3.1.1 Generation of INTACT Reporter Lines
3.1.2 Verification of the INTACT Reporter Lines
3.2 INTACT Procedure
3.2.1 Harvesting Starting Material
3.2.2 INTACT
3.2.3 Assessment of the Enrichment of Tissue-Specific Nuclei by Low-Input Smart-PCR
4 Notes
References
Part IV: Molecular Biology, Genomics, and Systems Biology
Chapter 17: RNA In Situ Hybridization on Plant Tissue Sections: Expression Analysis at Cellular Resolution
1 Introduction
2 Materials
2.1 Probe Synthesis and Dot Blot
2.2 Tissue Embedding and Sectioning
2.3 Probe Hybridization
2.4 Microscopy
3 Method
3.1 Probe Synthesis
3.2 Tissue Embedding and Sectioning
3.3 Probe Hybridization
3.4 Microscopy: The Secret of Good Picture-Taking
4 Notes
References
Chapter 18: The GUS Reporter System in Flower Development Studies
1 Introduction
2 Materials
2.1 Histochemical GUS Assay
2.1.1 GUS Staining
2.1.2 Embedding/Sectioning/Mounting
2.2 Fluorometric GUS Assay
2.2.1 Protein Extracts
2.2.2 Fluorometric Assay
3 Methods
3.1 Histochemical GUS Assay
3.1.1 GUS Staining
3.1.2 Embedding
3.1.3 Sectioning and Mounting
3.1.4 Alternative Simplified GUS Screening Assay
3.2 Fluorometric GUS Assay
3.2.1 Preparation of Protein Extracts
3.2.2 MU Standard Curve
3.2.3 MUG Assay on Plant Extracts
3.2.4 Bradford Assay
3.2.5 MUG Assay on Intact Inflorescences or Flowers
4 Notes
References
Chapter 19: Expression and Functional Studies of Leaf, Floral, and Fruit Developmental Genes in Non-model Species
1 Introduction
2 Materials
2.1 Small-Scale Expression Analyses
2.1.1 Plant Material Collection
2.1.2 Total RNA Isolation
2.1.3 RNA Quality Test
2.1.4 Sample Submission to the Sequencing Facilities
2.1.5 Primer Design
2.1.6 cDNA Synthesis
2.1.7 RT-PCR
2.1.8 Visualization and Result Readings
2.1.9 qRT-PCR
2.2 Virus-Induced Gene Silencing (VIGS)
2.2.1 Vector Construction
2.2.2 Plant Growth and Agroinfiltration
2.3 RNA-seq Data Analyses
2.4 Differentially Expressed Genes (DEGs) Identification
3 Methods
3.1 Experimental Design
3.2 Purification of Total RNA
3.2.1 Plant Material Collection
3.2.2 Total RNA Isolation
3.2.3 RNA Quality Test
3.2.4 Sample Submission to the Sequencing Facilities
3.3 RNA-seq Data Analyses
3.3.1 Read Quality Control and Preprocessing
3.3.2 Quality Trimming
3.3.3 Transcriptome Assembly
3.3.4 Orthologous Gene Identification
3.4 Differentially Expressed Genes (DEGs) Identification
3.4.1 Normalized Quantification of Transcripts from Reads with Kallisto (TPM)
3.4.2 Counts Generation
3.4.3 Results from Kallisto
3.4.4 Differential Gene Expression Analysis Using DESeq2
3.4.5 Figures on Differentially Expressed Genes (plotPCA)
3.5 Small Scale Expression Analyses
3.5.1 cDNA Synthesis
3.5.2 Primer Design
3.5.3 RT-PCR
3.5.4 Visualization and Analysis Results
3.5.5 qRT-PCR
3.6 Virus-Induced Gene Silencing
3.6.1 Vector Construction
3.6.2 Plant Growth and Agrobacterium Culture Preparation
3.6.3 Agroinfiltration
4 Notes
References
Chapter 20: Gene Expression Analysis by Quantitative Real-Time PCR for Floral Tissues
1 Introduction
2 Materials
2.1 Tissue Collection and RNA Extraction
2.2 Reverse Transcription Reaction
2.3 Quantitative Real Time PCR-LightCycler 480 System
2.4 Quantitative Real Time PCR-BioMark System
3 Methods
3.1 Tissue Collection and RNA Extraction
3.2 Reverse Transcription Reaction
3.3 Quantitative Real Time PCR: LightCycler 480 System
3.4 Quantitative Real Time PCR: BioMark System
3.5 Data Analysis
3.5.1 Absolute Quantification
3.5.2 Relative Quantification
4 Notes
References
Chapter 21: Misexpression Approaches for the Manipulation of Flower Development
1 Introduction
2 Materials
2.1 Construction of Expression Cassettes
2.2 Construction of CRISPR-Based Transcription Regulators
2.3 Agrobacterium-Mediated Transformation of Arabidopsis
2.4 Screening of Transgenic Plants
2.5 Dexamethasone Induction and Protein Synthesis Inhibition Treatment
2.6 Screening and Identification of Activation-Tagged Genes
2.7 Observation of Phenotypes
3 Methods
3.1 Construction of Expression Cassettes
3.1.1 Choice of Promoters
3.1.2 Choices of Inducible Systems
3.1.3 Cloning Step of Misexpression Vectors
3.1.4 Activation Tagging
3.2 Construction of CRISPR-Based Transcription Regulators
3.2.1 Preparation of CRISPR-Based Artificial Transcription Activator
3.2.2 Preparation of CRISPR-Based Artificial Transcription Repressor
3.3 Transformation of Arabidopsis (Agrobacterium-Mediated Transformation)
3.4 Screening of Transformants
3.5 Dexamethasone Induction and Protein Synthesis Inhibition Treatment
3.6 Screening and Determination of the Activation Tag
3.7 Observation of Phenotypes
4 Notes
References
Chapter 22: Genomic Approaches for the Study of Flower Development in Floriculture Crops
1 Introduction
1.1 Plant Material to Develop a Genome Draft Assembly
1.2 Plant Material Used to Develop a Mapping Population for the Phenotype of Interest
1.3 Plant Material Used for the Characterization of the Transcriptomic Landscape of the Phenotype of Interest
1.4 Genome Sequence Assembly
1.5 Genotyping and QTL Analysis of the Population
1.6 Transcriptomic Analysis
2 Materials
2.1 Generation of a Draft Genome Sequence
2.1.1 HMW DNA Extraction, Size Selection, and Size/Quality Evaluation
2.1.2 Library Preparation and DNA Sequencing
2.1.3 Base Calling and Genome Sequence Assembly
2.1.4 Genome Annotation
2.2 Genotyping and QTL Analysis of the Population
2.2.1 DNA Extraction and Quantification
2.2.2 Genotyping-By-Sequencing Library Preparation
2.2.3 Genotyping-By-Sequencing Data Processing
2.2.4 Genetic Map and QTL Analysis
2.3 Transcriptomic Analysis
2.3.1 RNA Extraction and Quality Evaluation
2.3.2 RNA-Seq Analysis
3 Methods
3.1 Generation of a Draft Genome Sequence
3.1.1 HMW DNA Extraction
3.1.2 Size Selection
3.1.3 DNA Quantification and Quality Evaluation
3.1.4 DNA Library Preparation
3.1.5 MinION DNA Sequencing and Base Calling
3.1.6 Genome Sequence Assembly
3.1.7 Genome Annotation
3.2 QTL Analysis
3.2.1 DNA Extraction
3.2.2 Genotyping-By-Sequencing Library Preparation
3.2.3 Genotyping-By-Sequencing Data Processing and Variant Calling
3.2.4 Development of the Genetic Map
3.2.5 QTL Analysis
3.3 Transcriptomic Analysis
3.3.1 RNA Extraction and Quality Evaluation
3.3.2 RNA-Seq Library Preparation
3.3.3 RNA-Seq Data Processing and Transcript Quantification
4 Notes
References
Chapter 23: Multi-Omics Methods Applied to Flower Development
1 Introduction
2 Materials
2.1 Protein Extraction
2.2 RNA Extraction
2.3 LC-MS/MS
3 Methods
3.1 Protein Extraction
3.2 RNA Extraction
3.3 LC-MS/MS
3.3.1 Sample Preparation
3.3.2 Chromatographic and Mass Spectrometric Analysis
3.3.3 Data Analysis
3.3.4 Treatment of Missing Values and Data Imputation
3.3.5 Example: Treatment of Missing Values in a Time Series Experiment
4 Notes
References
Chapter 24: Peptidomics Methods Applied to the Study of Flower Development
1 Introduction
2 Materials
2.1 General
2.2 Ultrafiltration
2.3 Ammonium Sulphate Precipitation
2.4 Reverse-Phase Chromatography Peptide Extraction
2.5 LC-MS/MS
3 Methods
3.1 Ultrafiltration
3.2 Ammonium Sulphate Precipitation
3.3 Reverse-Phase Chromatography Peptide Extraction
3.4 LC-MS/MS
3.4.1 Sample Preparation
3.4.2 Chromatographic and Mass Spectrometric Analysis
3.4.3 Data Analysis for Database-Search Peptide Identification
4 Notes
References
Chapter 25: Quantifying Gene Expression Domains in Plant Shoot Apical Meristems
1 Introduction
2 Method
2.1 Characterization of a Fluorescence Domain
2.2 Applications
2.2.1 Inflorescence Meristem Pipeline
2.2.2 Floral Meristems Pipeline
3 Notes
References
Chapter 26: A NanoLuc-Based Transactivation Assay in Plants
1 Introduction
2 Materials
2.1 NanoLuc and Overexpression Vectors
2.2 Agroinfiltration
2.3 NanoLuc Assay
2.4 Luminescence Detection
3 Methods
3.1 NanoLuc and Overexpression Vectors
3.2 Agroinfiltration
3.3 NanoLuc Assay in Plant Extracts
3.4 NanoLuc Assay in Entire N. benthamiana Leaves
4 Notes
References
Chapter 27: A Hands-On Guide to Generate Spatial Gene Expression Profiles by Integrating scRNA-seq and 3D-Reconstructed Micros...
1 Introduction
2 Materials
2.1 Computational Infrastructure
2.2 Required Datasets
3 Methods
3.1 Computational Environment Setup
3.1.1 Download Scripts and Data
3.1.2 Install Docker
3.1.3 Build the JupyterLab Docker Image
3.1.4 Start the JupyterLab Docker Image
3.1.5 Access the JupyterLab Docker Image
3.1.6 Stop the Docker Container
3.2 Predicting 3D Gene Expression Profiles and Cell-to-Cell Mappings
3.2.1 Loading Libraries and Setting Paths to the Input Data
3.2.2 Setting the Parameters
3.2.3 Loading the Required Data
3.2.4 Preprocessing the Data
3.2.5 Preparation for novoSpaRc-Based Expression Reconstruction
3.2.6 Predict Cell-to-Cell Mappings and 3D Gene Expression Profiles
3.2.7 Format and Save the Results
3.2.8 Visualizing 3D Gene Expression Profiles
3.3 Projecting scRNA-seq Clusters onto the 3D Meristem
3.4 Visualizing scRNA-seq Clusters in the 3D Flower Meristem
3.4.1 Load the Data
3.4.2 Visualize the UMAP Plot of scRNA-seq Cells
3.4.3 Plot the Projection of UMAP Cluster in the 3D Meristem
3.5 Evaluating Prediction Performance (AUCROC and PEP-Score)
4 Notes
References
Index