Preface
Contents
Contributors
Chapter 1: Circadian Rhythm: Phase Response Curve and Light Entrainment
1 Introduction
2 Materials
3 Methods
3.1 Preparing Arabidopsis Seedlings Culture Medium
3.2 Growth of Plant Seedlings in Sterile Conditions
3.3 Environmental Conditions in Experimental Setting and Light Entrainment
3.4 Preparing Luciferin for Bioluminescent Assays
3.5 Using Real-Time Reporter for Circadian Dynamics
3.6 Applying Light Pulses at all Possible Phases Across the Circadian Cycle
3.7 Determining Free-Running Period (Tau) and Circadian Time Phase
3.8 Determining Phase Response Characteristics and Plotting the Phase Response Curve
4 Notes
References
Chapter 2: Period Estimation and Rhythm Detection in Timeseries Data Using BioDare2, the Free, Online, Community Resource
1 Introduction
2 Materials
3 Methods
3.1 Creation of a New Experiment in BioDare2
3.2 Import of Numerical Data
3.3 Visualizing Timeseries
3.4 Period Estimation
3.4.1 Starting a New Analysis
3.4.2 Examining Results of Period Estimates
3.5 Rhythm Detection
3.5.1 Starting a New Rhythmicity Test
3.5.2 Examining Rhythmicity Test Results
4 Notes
References
Chapter 3: Detection and Analysis of Circadian Rhythms Via Prompt Chlorophyll Fluorescence
1 Introduction
2 Materials
2.1 System Setup
3 Methods
3.1 Determining Light Intensities for the Experiment
3.2 Plant Placement and Acclimation
3.3 Determining Measurement Areas
3.4 Protocol and Script Selection
3.5 Data Processing
3.6 Rhythm Analysis
3.6.1 Rhythm Analysis with BRASS
3.6.2 Rhythm Analysis with BioDare2
4 Notes
References
Chapter 4: High Spatial Resolution Luciferase Imaging of the Arabidopsis thaliana Circadian Clock
1 Introduction
2 Materials
3 Methods
3.1 Description of the Imaging Setup
3.2 Setting up a Time-Lapse Experiment
3.3 Analysis of Luciferase Images
4 Notes
References
Chapter 5: Agrobacterium-Mediated Seedling Transformation to Measure Circadian Rhythms in Arabidopsis
1 Introduction
2 Materials
2.1 Plant Growth
2.2 Agrobacterium Culture
2.3 Solutions
2.4 Vacuum Infiltration
2.5 Luminescence Detection
3 Methods
4 Notes
References
Chapter 6: Measurement of Luciferase Rhythms in Soybean Hairy Roots
1 Introduction
2 Materials
2.1 Plasmids, Vectors, Strains, and Plants
2.2 Media, Buffers, and Reagents
2.3 Apparatus and Accessories
3 Methods
3.1 Hairy Roots Transformation Using Agrobacterium rhizogenes K599
3.2 Endogenous Circadian Rhythm Measurement and Data Assay
4 Notes
References
Chapter 7: ODE (Ordinary Differential Equation) Models for Plant Circadian Networks: What the Models Are and How They Should B...
1 Fundamental Concepts: Models and ODEs
1.1 What Is a Circadian Model?
1.2 What Is an ODE? Some Mathematical Preliminaries
1.3 A Useful Example: Locke et al.
2 Qualitative Behavior of ODEs: Important Considerations for Circadian Biology
3 Solving ODE Models Computationally
4 Example MATLAB Code
References
Chapter 8: How to Detect QTLs in the Plant Circadian Clock
1 Introduction
2 Methods
2.1 QTL Mapping
2.1.1 Selection of Parental Accessions
2.1.2 Generation of Suitable Mapping Populations
2.1.3 Genotyping and Phenotyping
Genotyping
Phenotyping
2.1.4 Statistical Analysis to Detect QTLs
3 Notes
References
Chapter 9: Measuring Hypocotyl Length in Arabidopsis
1 Introduction
2 Materials and Experimental Considerations
2.1 Equipment and Software Required
2.2 Growth Conditions
2.3 Growth Media
3 Experimental Setup and Data Analysis
4 Notes
References
Chapter 10: High-Throughput Extraction and Enzymatic Determination of Sugars and Fructans in Fructan-Accumulating Plants
1 Introduction
2 Materials
2.1 Equipment and Consumables
2.2 Extraction of Soluble Compounds
2.3 Determination of Soluble Carbohydrates
2.4 Active Enzyme Solutions
3 Methods
3.1 Extraction of Soluble Carbohydrates (and Other Soluble Compounds)
3.2 Determination of Glucose, Fructose, Sucrose, and Fructans
3.2.1 Determination of Glucose, Fructose, and Sucrose
3.2.2 Denaturation of the Enzymes and NADH and Neutralization
3.2.3 Degradation of Fructans
3.2.4 Determination of Fructan-Derived Glucose and Fructose
3.3 Calculation
4 Notes
References
Chapter 11: Simple Nuclei Preparation and Co-immunoprecipitation Procedures for Studying Protein Abundance and Interactions in...
1 Introduction
2 Materials
2.1 Plants for Nuclei Purification and Co-IP
2.2 Reagents and Tools for Nuclei Purification
2.3 Reagents for Co-immunoprecipitation (Co-IP)
3 Methods
3.1 Isolation of Nuclei for Western Blot Analysis
3.2 Preparation of Plant Tissues Expressing Potential Interacting Proteins for Co-IP
3.3 Preparation of Antibody-Magnetic Bead Complex Prior to Co-IP
3.4 Whole-Cell Protein Extraction
3.5 Separate Cytosolic- and Nuclei-Enriched Fractions
3.6 Co-IP Procedure
4 Notes
References
Chapter 12: Chromatin Immunoprecipitation Protocol for Circadian Clock Proteins
1 Introduction
2 Materials
2.1 Plant Material
2.2 Equipment and Supplies
2.3 Solutions and Reagents
3 Methods
3.1 Experimental Design
3.1.1 Choice of Antibody
3.1.2 Control Samples
3.1.3 Control Loci
3.1.4 Biological Replicates
3.2 Protocol
3.2.1 Harvesting and Cross-Linking of Plant Tissue
3.2.2 Chromatin Isolation
3.2.3 Quality Control of the Chromatin and Isolation of the Input DNA Sample
3.2.4 Immunoprecipitation (Day 1)
3.2.5 Immunoprecipitation (Day 2)
3.2.6 Reversal of Cross-Links and DNA Purification
3.2.7 Alternative Protocol: Chelex Method
3.3 Downstream Analyses
3.3.1 Quantitative PCR
3.3.2 High-Throughput Sequencing
4 Notes
References
Chapter 13: A High-Throughput 3β²-Tag RNA Sequencing for Large-Scale Time-Series Transcriptome Studies
1 Introduction
2 Materials
2.1 Sampling of Plant Material and Grinding Tissues
2.2 RNA Extraction and Quality Assessment
2.3 Library Preparation
2.4 Analysis of 3β²-TagSeq Data
3 Methods
3.1 Sampling of Plant Material
3.2 Grinding
3.3 RNA Extraction
3.4 Quality Assessment of RNA
3.5 Library Preparation
3.5.1 RNA Fragmentation
3.5.2 First-Strand cDNA (FS-cDNA) Synthesis
3.5.3 cDNA Amplification
3.5.4 Sample-Specific Barcoded PCR
3.5.5 Pool Samples and Size Selection
3.5.6 Quantification for Mixing on the Same HiSeq2500 Lane
3.6 Bioinformatics Analysis of 3β²-TagSeq Data
3.6.1 Data Preparation
3.6.2 Read Quality Assessment
3.6.3 Read Filtering with Poly-A Tails and 5β² Adapters
3.6.4 Reference Index Building
3.6.5 Reads Mapping
3.6.6 Reads Counting
3.7 Statistical Analysis of Transcriptome Data
4 Notes
References
Chapter 14: Experimental Design for Time-Series RNA-Seq Analysis of Gene Expression and Alternative Splicing
1 Introduction
2 Materials
2.1 Seed Sterilization Solution (Arabidopsis)
2.2 Hydroponic Growth Solution
2.3 Isoform-Specific Primer Design for Expression/AS Validation
3 Methods
3.1 Arabidopsis Seed Sterilization and Stratification
3.2 Hydroponic Growth of Arabidopsis Plants
3.3 Time-Course Harvesting
3.4 RNA Isolation
3.5 Library Preparation and RNA Sequencing
3.6 RNA-Seq Analysis Pipeline
3.6.1 Validation of Gene and Transcript Expression with Reverse Transcription Quantitative PCR (RT-qPCR)
3.6.2 Validation of AS with High-Resolution RT-PCR
4 Note
References
Chapter 15: Using Tandem Affinity Purification to Identify Circadian Clock Protein Complexes from Arabidopsis
1 Introduction
2 Materials
2.1 Cross-Linking Antibodies to Protein G Beads
2.2 Plant Growth and Tissue Homogenization
2.3 Affinity Purification
3 Methods
3.1 Cross-linking Antibody to Protein G Beads (See Notes 5 and 6)
3.2 Plant Growth and Tissue Collection
3.3 Tissue Homogenization
3.4 Protein Extraction and Sonication (See Note 16)
3.5 Extract Clarification
3.6 Pre-wash Anti-FLAG Dynabeads
3.7 FLAG Immunoprecipitation
3.8 Bead Capture and Washes
3.9 Elution of Immunoprecipitated Proteins Off Anti-FLAG Beads
3.10 Wash His-Tag Isolation Dynabeads
3.11 His Immunoprecipitation and Washes
3.12 Quality Control Western Blot
4 Notes
References
Chapter 16: Firefly Luciferase Complementation-Based Analysis of Dynamic Protein-Protein Interactions Under Diurnal and Circad...
1 Introduction
2 Materials
2.1 Plasmids, Vectors, Strains, and Plants
2.2 Media, Buffers, and Reagents
2.3 Apparatus
3 Methods
3.1 Full-Length Gene Cloning, Vector Construction, and Transformation
3.2 Split-LUC Complementation Assay
4 Notes
References
Chapter 17: Monitoring Seasonal Bud Set, Bud Burst, and Cold Hardiness in Populus
1 Introduction
2 Materials
3 Methods
3.1 Propagation/Initiation
3.2 Growth
3.3 Winter Dormancy Measurements
3.3.1 Critical Day Length (CDL), Growth Cessation, and Bud Set
3.3.2 Freeze Test
3.3.3 Visual Score of Freeze Injury
3.3.4 Bud Burst
3.4 Statistical Processing of Data
3.5 Sampling of Material for Transcript or Protein Analysis
4 Notes
References
Chapter 18: The Perennial Clock Is an Essential Timer for Seasonal Growth Events and Cold Hardiness
Abbreviations
1 Introduction
2 Perennial Clocks
3 The Populus Clock
4 Seasonal Regulation by the Clock
5 Clocks in Other Perennial Plant Species
6 Seasonal Processes: Bud Set, Dormancy, and Bud Burst
7 The Role of Phytohormones in Seasonal Regulation
8 Temperature Effects in Dormancy
9 Outlook
References
Correction to: Period Estimation and Rhythm Detection in Timeseries Data Using BioDare2, the Free, Online, Community Resource
Index