Environmental responses in plants : methods and protocols.

Preface
Contents
Contributors
Part I: Tropisms and Other Developmental Responses
	Chapter 1: Analysis of Plant Root Gravitropism
		1 Introduction
		2 Materials
			2.1 General Materials
			2.2 Options for Image Acquisition
		3 Methods
			3.1 Preparing Plant Materials
			3.2 Example Data Processing Through SmartRoot and AstroDart
		4 Notes
		References
	Chapter 2: Hydrotropism: Analysis of the Root Response to a Moisture Gradient
		1 Introduction
		2 Materials
		3 Methods
			3.1 Growth of Arabidopsis Seedlings
			3.2 Preparation of Plates That Establish a Water Potential Gradient
			3.3 Root Curvature Measurement
		4 Notes
		References
	Chapter 3: Evaluating Root Mechanosensing Response in Rice
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Lab Material
			2.3 Imaging System (Optional)
		3 Methods
			3.1 Preparation of Plates with Yoshida Nutritional Medium (see Note 3)
			3.2 Surface Disinfection of Rice Seeds (see Note 4)
			3.3 Evaluation of Rice Root Mechanosensing Response (see Note 15 for Final Considerations and Remarks Regarding This Method)
			3.4 Imaging Rice Root Mechanical Response to Barrier (Optional)
		4 Notes
		References
	Chapter 4: Physiological Analysis of Phototropic Responses to Blue and Red Light in Arabidopsis
		1 Introduction
		2 Materials
		3 Methods
			3.1 Seed Preparation
			3.2 Germination and Light Treatments
			3.3 Measuring the Bending Angle with ImageJ
		4 Notes
		References
	Chapter 5: Shoot Branching Phenotyping in Arabidopsis and Tomato
		1 Introduction
		2 Materials
		3 Methods
			3.1 Scoring Branching in Arabidopsis
			3.2 Scoring Branching in Tomato
		4 Notes
		References
	Chapter 6: A Ribo-Seq Method to Study Genome-Wide Translational Regulation in Plants
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Consumables
			2.3 Reagents
			2.4 Enzymes
			2.5 Buffers and Solutions
			2.6 Oligonucleotides
		3 Methods
			3.1 Tissue Preparation
			3.2 Tissue Extract Preparation
			3.3 Separation of Monosomes from the RFP Lysates Using RNase I Digestion and Size-Exclusion Chromatography
				3.3.1 RNase Digestion of Polysomes in the Lysate
				3.3.2 Purification of Monosomes with Sephacryl S-400 Columns
			3.4 Purification of Ribosome Footprints
				3.4.1 Purification of RNA from the RFP Sample by Zymo RNA Clean -5 Kit
				3.4.2 Polyacrylamide Gel Electrophoresis and RFP RNA Purification
			3.5 Total RNA Fragment Purification
				3.5.1 Purification of RNA from Total RNA Supernatant by Zymo RNA Clean -5 Kit
				3.5.2 Fragmentation of Total RNA by Alkaline Lysis
				3.5.3 Size Selection and Purification of Total RNA Fragments
			3.6 Removal of 3′ Phosphoryl Groups from RFP and Total RNA Fragments
				3.6.1 PNK Treatment for RFP and Total RNA Fragments
				3.6.2 Purification of PNK-Treated RNA Fragments
			3.7 Ligation of the 3′ Adapters to RNA Fragments
			3.8 Purification of Ligated Products
			3.9 rRNA Depletion
				3.9.1 Removal of rRNA by Ribo-Zero Reagents
				3.9.2 Purification of rRNA-Depleted RNA
			3.10 Reverse Transcription
				3.10.1 Reverse Transcription of RNA into cDNA
				3.10.2 Size Selection and Purification of cDNA from Polyacrylamide Gel
			3.11 Circularization of cDNA
			3.12 Sequencing Library Preparation
				3.12.1 Library Amplification by PCR
				3.12.2 Gel Purification of the Library
				3.12.3 Library Quality and Quantity Measurement by Bioanalyzer
			3.13 Data Analysis in Brief
		4 Notes
		References
Part II: Photoperiodism and Circadian Rythms
	Chapter 7: Assessing Flowering Time Under Different Photoperiods
		1 Introduction
		2 Materials
		3 Methods
			3.1 Potting of Plant Material
			3.2 Thinning of Plant Material
			3.3 Scoring Flowering Time
				3.3.1 Scoring Flowering Time as Total Leaf Number
				3.3.2 Scoring Flowering Time as Days Until Bolting
			3.4 Data Analysis
			3.5 Examples of Flowering Time Scoring in Mutants
		4 Notes
		References
	Chapter 8: Analysis of Phytochrome-Dependent Seed Germination in Arabidopsis
		1 Introduction
		2 Materials
		3 Methods
			3.1 Seed Preparation
			3.2 Light Treatments
			3.3 Counting the Seeds
		4 Notes
		References
	Chapter 9: Rhythmic Leaf and Cotyledon Movement Analysis
		1 Introduction
		2 Materials
			2.1 Plant Materials
			2.2 Camera Apparatus
		3 Methods
			3.1 Planting
			3.2 Imaging
			3.3 Image Analysis (See Note 7) Using TRiP: A MATLAB-Based Program
		4 Notes
		References
	Chapter 10: Assessing Plant Pigment Regulation in Circadian Experiments
		1 Introduction
		2 Materials
			2.1 Growth Facility
			2.2 Materials for Noninvasive Techniques
				2.2.1 Devices
				2.2.2 Device Accessories
				2.2.3 Additional Materials
			2.3 Materials for Analytical Techniques
				2.3.1 Materials for Sampling
				2.3.2 HPLC Columns
				2.3.3 HPLC Mobile Phase
				2.3.4 Extraction Media
				2.3.5 Chromatography
		3 Methods
			3.1 Design of a Circadian Experiment
				3.1.1 Setting Up Conditions During Entrainment
				3.1.2 Setting Up Conditions During the Free-Run
				3.1.3 Starting Measurements
				3.1.4 Frequency of Sampling
				3.1.5 Number of Replicates
				3.1.6 Duration of Experiments
			3.2 Pigment Estimation by Noninvasive Methods
				3.2.1 Sample Preparation
				3.2.2 Fluorescence by Fluorometers
				3.2.3 Reflectance Measurements
				3.2.4 Reflectance Indexes Derived from Fixed Wavelength Instruments
			3.3 Pigment Quantitation by Analytical Methods
				3.3.1 Sample Collection
				3.3.2 Storage
				3.3.3 Sample Extraction and Preparation for  HPLC
				3.3.4 Sample Extraction and Preparation for Spectrophotometry
				3.3.5 Spectrophotometry. Equations Developed for Pigment Quantitation in Acetone Are (see Note 20) as Follows
				3.3.6 Chromatography
				3.3.7 Detection and Peak Integration
			3.4 Data Analyses
		4 Notes
		References
	Chapter 11: Chemical Perturbation of Chloroplast Ca2+ Dynamics in Arabidopsis thaliana Suspension Cell Cultures and Seedlings
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Plant Material
			2.3 Equipment
			2.4 Consumables
		3 Methods
			3.1 Screening the Chemicals in Cell Culture
			3.2 Validation of Selected Chemicals in Seedlings
		4 Notes
		References
Part III: Abiotic Stress Responses
	Chapter 12: Screening for Abiotic Stress Response in Rice
		1 Introduction
		2 Materials
			2.1 Materials and Instruments
			2.2 Preparation of Yoshida Nutrient Solution
			2.3 Preparation of Hydroponic Trays and Rice Seeds for Germination
				2.3.1 Construction of the Floats for Seedlings
				2.3.2 Seed Preparation
				2.3.3 Seedling Establishment
		3 Methods
			3.1 Evaluation of Salt Stress Response in Rice at the Seedling Stage
				3.1.1 Salt Stress Treatment
				3.1.2 Salt Stress Treatment for Molecular and Biochemical Analysis
				3.1.3 Recovery from Salt Stress Treatment
				3.1.4 Phenotyping and Sample Collection for Analysis
			3.2 Evaluation of Cold Stress Response in Rice at the Seedling Stage
				3.2.1 Seedling Preparation
				3.2.2 Cold Shock Assay (4 C)
				3.2.3 Cold Acclimation Assay (10 C  4 C)
				3.2.4 Recovery from Cold Treatments
				3.2.5 Phenotyping and Sample Collection for Analysis
			3.3 Evaluation of Drought Response in Rice at the Seedling Stage
				3.3.1 Drought Experimental Setup
				3.3.2 Preparation of the Potted Plants
				3.3.3 Drought Imposition
				3.3.4 Phenotyping and Sample Collection for Analysis
			3.4 Evaluation of Drought Simulation (PEG 6000 Method) in Rice at the Seedling Stage
				3.4.1 PEG-Induced Drought Stress and Sampling for Molecular and Biochemical Analysis
				3.4.2 Recovery and Phenotyping
			3.5 Evaluation of Submergence Response in Rice at the Seedling Stage
				3.5.1 Preliminary Germination Assay
				3.5.2 Submergence Setup and Control
				3.5.3 Evaluation of Rice Coleoptile Growth Under Submergence
		4 Notes
		References
	Chapter 13: Etiolated Hypocotyls: A New System to Study the Impact of Abiotic Stress on Cell Expansion
		1 Introduction
		2 Materials
			2.1 Seed Sterilization, Seed Plating, and Seedling Growth
				2.1.1 Plant Material
				2.1.2 Reagents and Solutions
				2.1.3 Consumables
				2.1.4 Equipment
			2.2 Seedling Transfer
				2.2.1 Plant Material
				2.2.2 Reagents and Solutions
				2.2.3 Consumables
				2.2.4 Equipment
			2.3 Image Acquisition and Hypocotyl Measurements
				2.3.1 Plant Material
				2.3.2 Equipment
		3 Methods
			3.1 Seed Sterilization
			3.2 Seed Plating
			3.3 Seedling Growth (Pre-treatment)
			3.4 Seedling Transfer
				3.4.1 ABA, High Salinity, and Drought Stress
				3.4.2 Cold and Heat Stress
			3.5 Recording Growth of Pre-treated Seedlings
			3.6 Recording Growth of Stress-treated and Untreated Seedlings
			3.7 Hypocotyl Measurements and Statistical Analysis
		4 Notes
		References
	Chapter 14: Assessing Postgermination Development in Arabidopsis thaliana Under Abiotic Stress
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Reagents and Solutions
			2.3 Consumables
			2.4 Equipment
		3 Methods
		4 Notes
		References
	Chapter 15: Assessing Abscisic Acid-Mediated Changes in Stomatal Aperture Through High-Quality Leaf Impressions
		1 Introduction
		2 Materials
			2.1 Plant Material
			2.2 Reagents and Solutions
			2.3 Consumables
			2.4 Imprinting Material
			2.5 Equipment
		3 Methods
			3.1 Plant Growth
			3.2 ABA Treatment
			3.3 Cotyledon Impressions
			3.4 Image Acquisition
			3.5 Data Analysis
		4 Notes
		References
	Chapter 16: Evaluation of the Anti-transpirant Activity of ABA Receptor Agonists in Monocot and Eudicot Plants
		1 Introduction
		2 Materials
			2.1 Plant Material and Culture
			2.2 Chemicals, Solutions, and Buffers
			2.3 Instruments
			2.4 Software
		3 Methods
			3.1 Evaluation of Anti-transpirant Activity in the Eudicot Model Arabidopsis thaliana Using Infrared Imaging
				3.1.1 Culture of Arabidopsis Plants for Thermo-Imaging Experiments
				3.1.2 Application of Anti-transpirants and Acquisition of Infrared Images
				3.1.3 Image Analysis and Leaf Temperature Calculation
			3.2 Evaluation of Anti-transpirant Activity in the Monocot C4 Model Setaria viridis Using Infrared Imaging
				3.2.1 Culture of Setaria viridis Plants for Thermo-Imaging Experiments
				3.2.2 Application of Anti-transpirants and Acquisition of Infrared Images
				3.2.3 Image Analysis and Leaf Temperature Calculation
		4 Notes
		References
	Chapter 17: Quantifying Phytohormones in Vivo with FRET Biosensors and the FRETENATOR Analysis Toolset
		1 Introduction
		2 Materials
		3 Methods
			3.1 Growing Seedlings for Microscopy
			3.2 Single Time Point Hormone Concentration Responses
			3.3 Hormone Treatment Time Course
			3.4 Microscopy
				3.4.1 Considerations
				3.4.2 Microscope Settings
			3.5 Image Analysis
				3.5.1 FRETENATOR Analysis Software Installation
				3.5.2 Segmentation and Ratiometric Image Analysis with the FRETENATOR Segment and Ratio Plugin
				3.5.3 Quantifying Emission Ratios in Different Tissues with the FRETENATOR ROI Labeller Plugin
		4 Notes
		References
	Chapter 18: Optical Imaging Resources for Crop Phenotyping and Stress Detection
		1 Introduction
		2 Imaging Resources
			2.1 Thermal Imaging
			2.2 Reflectance Sensing
			2.3 RGB Sensing
			2.4 Multispectral Imaging
			2.5 Hyperspectral Imaging
		3 Notes
		References
Part IV: Plant Interactions with Other Organisms
	Chapter 19: Analysis of Innate Immune Responses Against Pathogenic Bacteria in Arabidopsis, Tomato, and Barley
		1 Introduction
		2 Materials
			2.1 Plant Growth
			2.2 Media
			2.3 Bacteria
			2.4 Solutions
			2.5 Equipment
		3 Methods
			3.1 Plant Growth
				3.1.1 A. thaliana
				3.1.2 Tomato
				3.1.3 Barley
			3.2 Preparation of the Bacterial Inoculum
				3.2.1 Preparing the Inoculum for Inoculation by Infiltration
				3.2.2 Preparing the Inoculum for Inoculation by Spraying or Brushing
			3.3 Inoculation by Infiltration
				3.3.1 Arabidopsis thaliana
				3.3.2 Tomato
				3.3.3 Barley
			3.4 Inoculation by Spray Treatment
			3.5 Inoculation by Brushing
			3.6 Harvesting, Plating, and Analysis of the Infections
				3.6.1 Time Points for the Harvest of Bacteria
				3.6.2 Harvest of Bacteria in Silwet Buffer
				3.6.3 Determination of Bacterial Titers
		4 Notes
		References
	Chapter 20: Performing Infection Assays of Sporisorium reilianum f. sp. Zeae in Maize
		1 Introduction
		2 Materials
		3 Methods
			3.1 Cultivation of Maize
			3.2 Preparation of the S. reilianum Inoculum
			3.3 Infection of Maize Plants with S. reilianum
			3.4 Symptom Classification and Scoring
		4 Notes
		References
	Chapter 21: A Method to Measure the Damage Caused by Cell-Sucking Herbivores
		1 Introduction
		2 Materials
			2.1 Laboratorial Material
			2.2 Equipment
			2.3 Software
			2.4 Biological Material
		3 Methods
			3.1 Plant Rearing
			3.2 Spider Mite Cohort Creation
			3.3 Damage Induction
			3.4 Image Acquisition
			3.5 Primary Segmentation and Cropping in Fiji
			3.6 Training and Machine Learning Segmentation in ilastik
			3.7 Final Damage Quantification
			3.8 ilastik´s Error Correction
		4 Notes
		References
	Chapter 22: RNA Isolation from Nematode-Induced Feeding Sites in Arabidopsis Roots Using Laser Capture Microdissection
		1 Introduction
		2 Materials
			2.1 Seed Sterilization and Plant Growth
			2.2 Juvenile Hatching, Sterilization, and Inoculation
			2.3 Laser Capture Microdissection
			2.4 Tissue Embedding
			2.5 Cryosectioning
			2.6 OCT Removal from Microsections
		3 Methods
			3.1 Surface Sterilization of Arabidopsis Seeds
			3.2 Juvenile Hatching
			3.3 Juvenile Sterilization
			3.4 Infecting Arabidopsis Plants
			3.5 Laser Capture Microdissection
				3.5.1 Sample Collection, Fixation, and Cryo-Preservation
				3.5.2 OCT Embedding
				3.5.3 Cryosectioning
				3.5.4 OCT Removal and Microdissection
		4 Notes
		References
	Chapter 23: High-Throughput Profiling of Root-Associated Microbial Communities
		1 Introduction
		2 Materials
			2.1 Fractionation
			2.2 DNA Isolation
			2.3 DNA Quantification
			2.4 PCR I
			2.5 Primer Digestion
			2.6 PCR II
			2.7 Amplicon Visualization and Purification
				2.7.1 Purification of Total Amplicons
				2.7.2 Gel Purification of Selected Amplicons
			2.8 Amplicon Quantification
			2.9 Equimolar Pooling of Amplicons and Final Quantification
		3 Methods
			3.1 Fractionation
			3.2 DNA Isolation
			3.3 DNA Quantification
			3.4 PCR I
			3.5 Primer and dNTP Digestion
			3.6 PCR II
			3.7 Amplicon Purification
				3.7.1 Purification of Total Amplicons (See Note 9)
				3.7.2 Gel Purification of Selected Amplicons (Only for V5V7 and V2V4 16S rRNA Gene Libraries of in Planta Bacterial Communitie...
			3.8 Amplicon Quantification
			3.9 Equimolar Pooling of Amplicons and Final Quantification
		4 Notes
		References
	Chapter 24: Analysis of Immunity-Related Oxidative Bursts by a Luminol-Based Assay
		1 Introduction
		2 Materials
			2.1 Media
			2.2 Solutions and Chemicals
			2.3 Equipment
		3 Methods
			3.1 Plant Material
			3.2 Bacteria
			3.3 Sample Preparation
			3.4 Measurement
		4 Notes
		References
Index