Plant Photomorphogenesis: Methods and Protocols (Methods in Molecular Biology, 2297)

✍ Scribed by Ruohe Yin (editor), Ling Li (editor), Kaijing Zuo (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 181
• Category: Library

✦ Synopsis

This book provides detailed protocols for research in plant photomorphogenesis. The collection includes a broad range of topics including assays for shade avoidance responses, assays for light-dependent protein-protein interactions, photobody detection with immunofluorescence and the super-resolution imaging method, protein complex isolation from plants, detection of homodimer and monomer of photoreceptor UVR8 with immunoblotting analysis, assays for seedling greening, procedures for studying skotomorphogenesis, phenotypic study of photomorphogenesis at the seedling stage, expression of Cryptochrome in insect cells, and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Plant Photomorphogenesis: Methods and Protocols serves as an ideal guide for researchers and students who are new to the field, as well as a stepping stone for experienced researchers to further their skills in this fast-developing field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Setting Up an Arabidopsis LED Culture Module that Simulates Plant Neighbor Proximity
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 2: Photobody Detection Using Immunofluorescence and Super-Resolution Imaging in Arabidopsis
1 Introduction
2 Materials
2.1 Equipment
2.1.1 Equipment for Sample Preparation
2.1.2 Equipment for Imaging
2.2 Plant Material
2.3 Reagents and Solutions for Nuclei Isolation
2.4 Reagents and Solutions for Immunostaining and RNA FISH
2.5 Reagents and Solutions for DNA Staining
3 Methods
3.1 Isolation of Nuclei from Arabidopsis Leaf Tissue
3.2 Immunolabeling of Isolated Nuclei
3.2.1 Antibodies
3.2.2 Antibody Incubation
3.2.3 Detection and Staining
3.3 RNA Fluorescence In Situ Hybridization (FISH)
3.3.1 In Situ Hybridization
3.3.2 Detection and Imaging
3.4 Super-Resolution Imaging of Nuclear Photobodies in Arabidopsis
4 Notes
References
Chapter 3: Analysis of Shade-Induced Hypocotyl Elongation in Arabidopsis
1 Introduction
2 Materials
3 Methods
3.1 MS/2 Plates Preparation
3.2 Seeds Sterilization and Plating
3.3 Growth Incubator Preparation and Seedlings De-etiolation
3.3.1 Germination and De-etiolation
3.4 Shade Treatment
3.4.1 Neighbor Detection (Low R/FR Ratio)
3.4.2 Low Blue (Low B)
3.4.3 True Shade (Low B and Low R/FR Ratio)
3.5 Hypocotyl Measurements
4 Notes
References
Chapter 4: Isolation of UVR8 Protein Complexes
1 Introduction
2 Materials
2.1 Seed Sterilization
2.2 Protein Extraction
2.3 Beads Washing and Elution
3 Methods
3.1 Preparation of Plant Materials
3.2 Extraction of Total Proteins
3.3 Prewashing of Beads
3.4 Co-immunoprecipitation
3.5 Washing and Eluting Protein from Beads
4 Notes
References
Chapter 5: Phenotypic Study of Photomorphogenesis in Arabidopsis Seedlings
1 Introduction
2 Materials
2.1 Plant Culture
2.2 Measurement Physiological Parameters
3 Methods
3.1 Growth Condition
3.2 Set up the Monochromatic Light
3.3 Measurement Hypocotyl Length of Seedlings
3.4 Measurement Cotyledon Size of Seedlings
3.5 Measurement Anthocyanin Content of Seedlings
4 Notes
References
Chapter 6: Experimental Procedures for Studying Skotomorphogenesis in Arabidopsis thaliana
1 Introduction
2 Materials
2.1 Solutions for Phenotypic Analysis
2.2 Materials for RNA Isolation
2.3 Reagents for Reverse Transcriptional PCR
2.4 Solutions for qRT-PCR
2.5 Materials for Protein Extraction
2.6 Instruments
3 Method
3.1 Phenotypic Analysis
3.2 RNA Isolation
3.3 Removal of DNA from RNA Samples Prior to Reverse Transcriptional PCR
3.4 Reverse Transcription
3.5 qRT-PCR Analysis
3.6 Protein Extraction
3.7 Hormone Interaction Analysis
4 Notes
References
Chapter 7: Global Identification for Targets of Circadian Transcription Factors in Arabidopsis and Rice Using Chromatin Immuno...
1 Introduction
2 Materials
2.1 Plant Tissue Fixation
2.2 Chromatin Isolation
2.3 Immunoprecipitation
2.4 Reverse Cross-Linking
2.5 ChIP-seq Library Construction
3 Methods
3.1 Experimental Design
3.2 Fixation of Plant Material
3.3 Chromatin Isolation
3.4 Check Sonication Efficiency
3.5 Washing and Blocking the Beads, Pre-clearing the Sonicated Chromatin
3.6 Immunoprecipitation
3.7 Reverse Cross-Linking
3.8 ChIP-seq Library Construction
3.9 ChIP-seq Data Analysis
4 Notes
References
Chapter 8: Co-immunoprecipitation Assays to Detect In Vivo Association of Phytochromes with Their Interacting Partners
1 Introduction
2 Materials
2.1 Stock Solution
2.2 Pre-lysis Buffer
3 Methods
3.1 Protein Extraction
3.2 Immunoprecipitation
3.3 Purification of Immune Complexes
3.4 Immunoblot Analysis
4 Notes
References
Chapter 9: Detection of UVR8 Homodimers and Monomers by Immunoblotting Analysis in Tomato
1 Introduction
2 Materials
2.1 Technical Equipment
2.2 Buffers and Solutions
2.3 Plant Materials and Gene Identifier
2.4 Antigens
3 Methods
3.1 Sample Preparation
3.2 Gel Electrophoresis
3.3 UV-B Treatment of Protein Gels
3.4 Protein Transfer
3.5 Blocking the Membrane
3.6 Antibody Incubation
3.7 Imaging and Data Analysis
4 Notes
References
Chapter 10: Characterization of Seedling Greening Process in Plant Photomorphogenesis
1 Introduction
2 Materials
2.1 Plant Materials and Growth Conditions
2.2 Reagents and Equipments
2.2.1 Common Solutions
2.2.2 Seed Sterilization Solution and MS Medium
2.2.3 Pchlide Content Analysis
2.2.4 Cell Death Detection
Trypan Blue Staining
Electrolyte Leakage Measurement
2.2.5 Reactive Oxygen Species Measurement
Diaminobenzidine (DAB) Staining
Nitroblue Tetrazolium (NBT) Staining
Singlet Oxygen Fluorescence
H2DCFDA Fluorescent Dye
3 Methods
3.1 Greening Rate Measurement
3.2 Pchlide Content Analysis
3.3 Cell Death Detection
3.3.1 Trypan Blue Staining
3.3.2 Electrolyte Leakage Measurement
3.4 ROS Histochemical Staining
3.4.1 DAB Staining
3.4.2 NBT Staining
3.4.3 Singlet Oxygen Fluorescence Determination
3.4.4 ROS Fluorescence Determination
4 Notes
References
Chapter 11: Protoplast System for Studying Blue-Light-Dependent Formation of Cryptochrome Photobody
1 Introduction
2 Materials
2.1 Plant Cultivation
2.2 Solutions for Isolation of Arabidopsis Mesophyll Protoplasts
2.2.1 Stock Solution
2.3 Solution for Isolation of Soybean Mesophyll Protoplasts
2.4 Transient Transfection
3 Methods
3.1 Isolation and Transfection of Arabidopsis Mesophyll Protoplast
3.2 Isolation and Transfection of Soybean Mesophyll Protoplast
3.3 Detecting the Formation of Cryptochrome Photobodies
4 Notes
References
Chapter 12: Uncover the Nuclear Proteomic Landscape with Enriched Nuclei Followed by Label-Free Quantitative Mass Spectrometry
1 Introduction
2 Materials
2.1 The Plant Nuclei Isolation/Extraction Kit
2.2 Extraction Buffer
2.3 Preparation of SDS-PAGE Gel
2.4 Equipment
2.5 Reagents
2.6 Software
3 Methods
3.1 Cell Lysis
3.2 Semi-Pure Preparation of Nuclei
3.3 Nuclear Protein Extraction
3.4 Reduce the Nuclear Proteins and Block Cysteine
3.5 Digest the Proteins with Trypsin
3.6 Label-Free Protein Quantitation Analysis Using SWATH Method
3.6.1 Spectral Library Building
3.6.2 Quantitative Analysis Using SWATH Method
4 Notes
References
Chapter 13: Strategies to Study Dark Growth Deficient or Slower Mutants in Chlamydomonas reinhardtii
1 Introduction
2 Materials
2.1 Glassware
2.2 Compostable Materials
2.3 Apparatus
2.4 Chemicals and Reagents
3 Methods
3.1 Tris-Acetate-Phosphate (TAP) Medium Preparation
3.2 Growth Conditions
3.3 Phenotypic Analyses of the Mutants on TAP Agar Plates
3.4 Phenotype Analyses of the Mutants in TAP Liquid Media
3.5 Measurement of Oxygen Consumption and Evolution Rate Under Dark Heterotrophic Conditions
3.6 Analysis of Mitochondrial Protein Localization Under Dark Heterotrophic Conditions
3.7 Quantification and Qualification of Metabolites Under Dark Heterotrophic Conditions
3.8 Measurements of the Chlorophyll Fluorescence Parameters Under Dark Heterotrophic Conditions
3.9 Determination of Middle Point Redox Potential Under Dark Heterotrophic Conditions
4 Notes
References
Chapter 14: Co-immunoprecipitation Assay for Blue Light-Dependent Protein Interactions in Plants
1 Introduction
2 Materials
2.1 Put out Seeds on Plates
2.2 Co-immunoprecipitation
3 Methods
3.1 Put out Seeds on Plates
3.2 Grow and Collect Seedlings
3.3 Co-immunoprecipitation
4 Notes
References
Chapter 15: Detecting Blue Light-Dependent Protein-Protein Interactions by LexA-Based Yeast Two-Hybrid Assay
1 Introduction
2 Materials
2.1 Plasmids and Yeast Strain
2.2 Stock Solution
2.3 Media
2.4 Buffers and Reagents
2.5 Equipment
3 Methods
3.1 Preparation of Competent Yeast Cells
3.2 Transformation of Competent Yeast Cells
3.3 Analysis of the Positives
4 Notes
References
Chapter 16: Express Arabidopsis Cryptochrome in Sf9 Insect Cells Using the Baculovirus Expression System
1 Introduction
2 Materials
2.1 Prepare Recombinant Bacmid and Baculovirus
2.2 Purify Proteins
3 Method
3.1 Generating the Recombinant Bacmid
3.2 Producing the Recombinant Baculovirus
3.3 Expressing and Purifying the Recombinant Protein
4 Notes
References
Chapter 17: Semi-In-Vivo Pull-Down Assay for Blue Light-Dependent Protein Interactions
1 Introduction
2 Materials
2.1 Prepare Proteins
2.2 Make SDS-PAGE Gel
2.3 Western Blot
3 Method
3.1 Generating Recombinant Proteins
3.2 Semi-In-Vivo Pull-Down Assay
3.3 SDS/PAGE and Immunoblot
4 Notes
References
Chapter 18: Tobacco System for Studying Protein Colocalization and Interactions
1 Introduction
2 Materials
2.1 Agrobacteria Transformation
2.2 Infiltration
2.3 Confocal Microscopy and CoIP
3 Methods
3.1 Transform Agrobacteria and Make Liquid Culture (See Note 3)
3.2 Adjust OD600
3.3 Infiltration
3.4 Confocal Microscopy for Protein Colocalization
3.5 CoIP
4 Notes
References
Index