Plant gravitropism : methods and protocols

Preface
Contents
Contributors
Chapter 1: Plant Gravitropism: From Mechanistic Insights into Plant Function on Earth to Plants Colonizing Other Worlds
	1 Introduction
	2 Brief Overview of Gravitropism and Insights from Omics Studies
	3 Evolution of Fast Gravitropism
	4 New Insights on the Role of Auxin and Other Hormones in Gravitropism
	5 Insights on Gravitropism and Other Plant Movements Enabled by Research in Microgravity
	6 Gravitropism for Optimizing Resource Acquisition Efficiency in Crop Plants
	7 Conclusions
	References
Chapter 2: Evaluation of Gravitropism in Non-seed Plants
	1 Introduction
	2 Materials
		2.1 Preparation of BCD Media
		2.2 Preparation of  MS Media and Materials for Tissue Sterilization
		2.3 Root Scanning
	3 Methods
		3.1 Cultivation of Physcomitrium patens
		3.2 Cultivation of Lycophyte Selaginella Moellendorffii for Gravitropic Analysis
		3.3 Cultivation of Fern Ceratopteris richardii for Gravitropic Analysis
		3.4 Set up of the Vertical Scanner System Used for Gravitropism Analysis
		3.5 Statistical Analysis of the Gravitropism of Non-seed Plant Species
	4 Notes
	References
Chapter 3: Spore Preparation and Protoplast Isolation to Study Gravity Perception and Response in Ceratopteris richardii
	1 Introduction
	2 Materials
		2.1 Sterilization and Synchronization of Spores
		2.2 Spore Culture Preparation
		2.3 Imaging Spores for Rhizoid Orientation and Analysis
		2.4 Protoplast Isolation from Ceratopteris Gametophytes
	3 Methods
		3.1 Spore Preparation
		3.2 Spore Culture Preparation
		3.3 Evaluating Rhizoid Orientation to Determine Direction of Polarization
		3.4 Protoplast Isolation from Ceratopteris Gametophytes
	4 Notes
	References
Chapter 4: A Multiplexed, Time-Resolved Assay of Root Gravitropic Bending on Agar Plates
	1 Introduction
	2 Materials
		2.1 Plant Growth Medium
		2.2 Automatic Scanning System
		2.3 Data Analysis
	3 Methods
		3.1 Plant Growth Media Preparation
		3.2 Preparation of Seedlings
		3.3 Transfer Seedlings to Assay Plates
		3.4 Scanning Root Bending Using an Automated Scan Program
		3.5 Root Angle Analysis
	4 Notes
	References
Chapter 5: Methods to Quantify Cell Division and Hormone Gradients During Root Tropisms
	1 Introduction
	2 Materials
		2.1 Quantifying Cell Division
			2.1.1 EdU Staining
			2.1.2 GUS Staining, Cell Counting, and Hormone Gradient Determination
	3 Methods
		3.1 Orientation Determination During Imaging of Tropic-Treated Roots
		3.2 EdU Staining and Fluorescence Intensity Measurements
		3.3 GUS Staining and Signal Quantification
		3.4 Cell Numbers in the Root Meristem Region
		3.5 Hormone Gradients Quantification
	4 Notes
	References
Chapter 6: Using the Automated Botanical Contact Device (ABCD) to Deliver Reproducible, Intermittent Touch Stimulation to Plan...
	1 Introduction
	2 Materials
		2.1 Construction of the  ABCD
		2.2 Arabidopsis Media
		2.3 Arabidopsis Pots, Flats, and  Soil
	3 Methods
	4 Notes
	References
Chapter 7: High-Resolution Kinematic Analysis of Root Gravitropic Bending Using RootPlot
	1 Introduction
	2 Materials
	3 Methods
		3.1 Arabidopsis Seed Germination
		3.2 Transfer of Arabidopsis seedlings into Experimental Chambers
		3.3 Vertical Stage Microscopy of Root Growth and Gravitropism
		3.4 Tracking Root Growth
		3.5 Kinematic Analysis of Root Growth Using RootPlot_v1
			3.5.1 Root Growth  Rate
			3.5.2 Root Angle
			3.5.3 Root Velocity Profile
			3.5.4 Root REGR (=Strain) Profile
	4 Notes
	References
Chapter 8: Staging of Emerged Lateral Roots in Arabidopsis thaliana
	1 Introduction
	2 Materials
		2.1 Plant Growth Medium and Seedling Growth
		2.2 Specimen Preparation
		2.3 Imaging and Analysis
	3 Methods
		3.1 Plant Material Growth
		3.2 Specimen Preparation
		3.3 Image Acquisition
		3.4 LR Classification
	4 Notes
	References
Chapter 9: Methods for a Quantitative Comparison of Gravitropism and Posture Control Over a Wide Range of Herbaceous and Woody...
	1 Introduction
	2 Materials
		2.1 Preparation and Imaging of Plants
		2.2 Software
	3 Methods
		3.1 Preparation and Imaging of Plants
		3.2 Image Analysis and Semi-Automatic Estimation of Parameters
	4 Notes
	References
Chapter 10: The Analysis of Gravitropic Setpoint Angle Control in Plants
	1 Introduction
	2 Materials
		2.1 Lateral Root Growth Angle and Reorientation Assays
		2.2 Lateral Shoot Branch Growth Angle and Reorientation Assays
		2.3 Clinorotation of Lateral Root and Shoot Branches
		2.4 Reorientation Kinetics of Lateral Roots
		2.5 Lateral Root Growth Angle and Reorientation Assays in Larger Seedlings
	3 Methods
		3.1 Lateral Root Reorientation Assays
		3.2 Lateral Shoot Branch Reorientation Assays
		3.3 Quantification of Lateral Root Growth Angle Profiles
		3.4 Quantification of Lateral Shoot Branch Growth Angle Profiles
		3.5 Clinorotation of Lateral Root Branches
		3.6 Clinorotation of Lateral Shoot Branches
		3.7 Reorientation Kinetics of Lateral Roots
		3.8 Lateral Root Growth Angle and Reorientation Assays in Larger Seedlings
	4 Notes
	References
Chapter 11: A Flat Embedding Method to Orient Gravistimulated Root Samples for Sectioning
	1 Introduction
	2 Materials
	3 Methods
		3.1 Preparation of Glass Slides
		3.2 Coating Glass Slides with Liquid Release Agent
		3.3 Flat Embedding Procedure
		3.4 Extracting Specimens from Flat Embedded Resin
	4 Notes
	References
Chapter 12: Conducting Plant Experiments in Space and on the Moon
	1 Importance of Plant Space Biology
	2 Principal Methods of Spaceflight Research
	3 Our Experience with Spaceflight Research with Plants
	4 How Do You Get an Experiment to Fly in Space?
	5 Three Narratives for Implementation of Spaceflight Experiments
		5.1 Biorack
		5.2 EMCS
		5.3 BRIC-16
	6 New Approaches to Genomics Research
		6.1 NASA GeneLab Project
		6.2 TOAST AstroBotany Database
		6.3 Moving from DNA Microarrays to RNA-seq Methods
	7 Challenges of Space Research
		7.1 Logistical Issues
		7.2 Development of Spaceflight Hardware and Procedures
		7.3 Resource Constraints in Spaceflight Experiments
		7.4 Organizational Issues with NASA and Other Space Agencies
		7.5 Interpretation and Publishing Data from Spaceflight Experiments
	8 Lessons Learned
	9 Outlook and Future Prospects
	References
Chapter 13: Plant Proteomic Data Acquisition and Data Analyses: Lessons from Spaceflight
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Equipment
		2.3 Extraction of Soluble Protein
		2.4 Extraction of Microsomal Membrane Proteins
		2.5 Proteomic Analysis
	3 Methods
		3.1 Plant Material
		3.2 Extraction of Microsomal Membrane Proteins
		3.3 Carbonate  Wash
		3.4 Extraction of Soluble Proteins
		3.5 Proteomic Analysis
		3.6 MS/MS Data Analysis
		3.7 Differential Abundance Analysis (See Note 21)
		3.8 Network-Based Proteomics
		3.9 Studying Unknown Proteins of Interest
	4 Notes
	References
Chapter 14: Blueprints for Constructing Microgravity Analogs
	1 Introduction
	2 Materials
		2.1 Support Structure
		2.2 Drives
		2.3 Power System and Electronics
	3 Methods
		3.1 Frame Assembly
		3.2 Attachment of the Motors
		3.3 Power Considerations
		3.4 Assembly of Electrical Components
		3.5 Adjustments of the Stepper Drivers
		3.6 Illumination
			3.6.1 Light Emitting Diodes (LEDs)
			3.6.2 LED Connection and Power Considerations
		3.7 Bill of Materials
		3.8 Assembly Instructions
			3.8.1 Frame
			3.8.2 Power Box Assembly
		3.9 Programming
		3.10 Accelerometer
		3.11 Potential Experiments
			3.11.1 Randomized, Complete Rotation
			3.11.2 Contrasting Complete Rotation with Fractional Rotations
			3.11.3 Asymmetrical Rotation
			3.11.4 Light Effects
	4 Notes
	References
Chapter 15: Arabidopsis Growth and Dissection on Polyethersulfone (PES) Membranes for Gravitropic Studies
	1 Introduction
	2 Materials
	3 Methods
		3.1 Guar Gum Preparation
		3.2 Seed Sterilization
		3.3 Plating Seeds
		3.4 Hydration and Growth
		3.5 Tissue Dissection
	4 Notes
	References
Chapter 16: Use of Reduced Gravity Simulators for Plant Biological Studies
	1 Introduction
		1.1 Simulated Microgravity on Earth: Concept and Limitations
		1.2 Simulating the Moon and Mars g-Levels on Earth
		1.3 Mechanical Versus Magnetic Levitation Facilities
		1.4 General Hardware Constraints: Comparison with Spaceflight Constraints
	2 Materials
		2.1 Ground-Based Facilities to Simulate Microgravity
		2.2 Seedling Growth and Cell Cultures
	3 Methods
		3.1 Use of Seedlings on Mechanical Facilities
			3.1.1 Use of Seedlings on Agar-Based Substrates
			3.1.2 Use of Seedlings on Paper-Based Substrates
		3.2 Use of Seedlings on Magnetic Levitation Facilities
		3.3 Use of Callus Cell Cultures on Mechanical and Magnetic Levitation Facilities
		3.4 Use of Cell Suspension Cultures on Mechanical and Magnetic Levitation Facilities
	4 Notes
	References
Chapter 17: Understanding the Mechanisms of Gravity Resistance in Plants
	1 Introduction
	2 Materials
		2.1 Plant Cultivation
		2.2 Fixation and Storage of Plant Materials
		2.3 Analysis of the Cell Wall Properties
		2.4 Microscopic Analysis of Plant Materials
	3 Methods
		3.1 Plant Cultivation
		3.2 Fixation and Storage of Plant Materials
		3.3 Analysis of the Cell Wall Properties
		3.4 Microscopic Analysis of Plant Materials
	4 Notes
	References
Chapter 18: NASA´s Ground-Based Microgravity Simulation Facility
	1 Introduction
	2 Research Retrospective
		2.1 Plant Research
		2.2 Non-Plant Research
	3 Gaps in Ground-Based Microgravity Simulation Research
	4 KSC Ground-Based Microgravity Simulation Support Capabilities
		4.1 Using Multiple Microgravity Research Platforms and High Fidelity Controls
		4.2 Minimizing Mechanical and Design Artifacts
		4.3 Computational Fluid Dynamic Models
		4.4 Capabilities for Designing Experiment Unique Equipment Add-ons
			4.4.1 General Considerations for Designing Hardware to Accommodate Plant Research
			4.4.2 Customized Plant Growth Systems
			4.4.3 Microgreen Plant Growth Chamber
			4.4.4 Live Cell Imagery under Simulated Microgravity Conditions
	5 Future Work to Support Science Beyond Low Earth Orbit
	References
Chapter 19: Evaluating the Effects of the Circadian Clock and Time of Day on Plant Gravitropic Responses
	1 Introduction
		1.1 The Importance of Time in Experimental Design
		1.2 The Time of Day Variation in Control Conditions
		1.3 Circadian Gating of Responses
		1.4 What Time of the Day Should an Experiment Be Performed?
		1.5 Comparing Results Between Experiments
	2 Materials
		2.1 Plant Growth
		2.2 Time Course and IR Imaging
	3 Methods
		3.1 Plant Growth Basics
		3.2 Seed Surface Sterilization and Plating
		3.3 Raspberry Pi Computer Set-up
		3.4 Gravistimulation Time Course
		3.5 Measuring Root Growth Response
		3.6 Evaluating for Time of Day or Circadian-Specific Responses
		3.7 Simultaneously Sampling Multiple Time points on the  RPM
		3.8 Visualization and Analysis of Time Course  Data
	4 Notes
	References
Index