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THÔNG TIN TÀI LIỆU
Cấu trúc
Preface
Contents
Contributors
Part I: Analysis of Ethylene Biosynthesis
Chapter 1: Gas Chromatography-Based Ethylene Measurement of Arabidopsis Seedlings
1 Introduction
2 Materials
2.1 Sterilize Arabidopsis Seeds
2.2 Preparation of Headspace GC Vials with Arabidopsis Seeds
2.3 Measurement of Ethylene with a Gas Chromatograph
3 Methods
3.1 Surface Sterilize Arabidopsis Seeds
3.2 Preparation of Headspace GC Vials with Arabidopsis Seeds
3.3 Measurement of Ethylene with a Gas Chromatograph
3.4 Calculation of Ethylene Concentration from the Samples
4 Notes
References
Chapter 2: Plant Ethylene Detection Using Laser-Based Photo-�Acoustic Spectroscopy
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Measurements
3.2 Calibration of the ETD
3.3 Flushing the ETD
3.4 Measurements in Continuous Flow Mode
3.5 Data Analysis in Continuous Flow Mode
3.6 Measurements in Stop-and-Flow Mode
3.7 Data Analysis in Stop-and-Flow Mode
3.8 Measurements in Samples Mode
3.9 Data Analysis in Samples Mode
4 Notes
References
Chapter 3: Treatment of Plants with Gaseous Ethylene and Gaseous Inhibitors of Ethylene Action
1 Introduction
2 Materials
2.1 Closed System for Ethylene Treatment
2.2 Ethephon as an Ethylene Source
2.3 1-MCP Treatment to Inhibit Ethylene Action
2.4 2,5-NBD Treatment to Inhibit Ethylene Action
2.5 Flow-Through System for Ethylene Treatment
3 Methods
3.1 Closed System for Ethylene Treatment
3.2 Ethephon as an Ethylene Source
3.3 1-MCP Treatment to Inhibit Ethylene Action
3.4 2,5-NBD Treatment to Inhibit Ethylene Action
3.5 Flow-Through System for Ethylene Treatment
4 Notes
References
Chapter 4: Analysis of 1-Aminocyclopropane-1-Carboxylic Acid Uptake Using a Protoplast System
1 Introduction
2 Materials
2.1 Isolation of Mesophyll Protoplasts from Arabidopsis Leaf
2.2 [14C]ACC Uptake Assay
3 Methods
3.1 Isolation of Mesophyll Protoplasts from Arabidopsis Leaf
3.2 [14C]ACC Uptake Assay
4 Notes
References
Chapter 5: Escherichia coli-Based Expression and In Vitro Activity Assay of 1-Aminocyclopropane-1-Carboxylate (ACC) Synthase and ACC Oxidase
1 Introduction
2 Materials
2.1 Construction of Expression Vectors
2.1.1 ACC Synthase
2.1.2 ACC Oxidase
2.2 Protein Expression
2.2.1 ACC Synthase
2.2.2 ACC Oxidase
2.3 Extraction and Purification
2.3.1 ACC Synthase
2.3.2 ACC Oxidase
2.4 Quality Check of Expressed Protein
2.5 Assay of ACC Synthase Activity
2.6 Assay of ACC Oxidase Activity
3 Methods
3.1 Construction of Expression Vectors
3.1.1 ACC Synthase
3.1.2 ACC Oxidase
3.2 Protein Expression
3.2.1 ACC Synthase
3.2.2 ACC Oxidase
3.3 Extraction and Purification
3.3.1 ACC Synthase
3.3.2 ACC Oxidase
3.4 Quality Check of Expressed Protein
3.5 Assay of ACC Synthase Activity
3.5.1 Enzyme Reaction
3.5.2 Quantify ACC by the Method of Lizada and Yang [15]
3.6 Assay of ACC Oxidase Activity
4 Notes
References
Chapter 6: Assay Methods for ACS Activity and ACS Phosphorylation by MAP Kinases In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Reagents and Kits
2.2 Buffers Used in Kinase and ACS Activity Assay
2.3 Equipment
3 Methods
3.1 Preparation of Recombinant Proteins
3.2 Activation of Recombinant MAPKs Using Constitutively Active Upstream MAPKKs
3.3 Phosphorylation of ACS Protein Using Activated Recombinant MAPKs
3.4 Phosphorylation of ACS Protein Using Immunoprecipitated Native MAPKs (Immune-Complex Kinase Assay)
3.5 Quantification of Phospho-ACS Protein Using Liquid Scintillation Counting
3.6 Quantification of Phospho-ACS Protein Using SDS-PAGE
3.7 ACS Activity Assay of Recombinant ACS Protein
3.8 ACS Activity Assay of Native ACS Protein in Total Protein Extracts or After Immuno-precipitation (Immune-Complex ACS Assay)
3.9 Detection of In Vivo Phosphorylation of Endogenous ACS Proteins
4 Notes
References
Part II: Analysis of the Ethylene Signaling Pathway
Chapter 7: Analysis of Ethylene Receptors: Ethylene-Binding Assays
1 Introduction
2 Materials
2.1 Growing and Isolating Intact Yeast
2.2 Isolating Yeast Membranes.
2.3 Solubilizing Membranes
2.4 Ethylene-Binding Assay
2.5 Preparation of Mercuric Perchlorate Solution for Trapping Ethylene
3 Methods
3.1 Isolating Intact Yeast
3.2 Isolating Yeast Membranes
3.3 Solubilizing Membranes
3.4 Ethylene-Binding Assay
3.5 Preparation of Mercuric Perchlorate Solution for Trapping Ethylene
4 Notes
References
Chapter 8: Analysis of Ethylene Receptors: Assay for Histidine Kinase Activity
1 Introduction
2 Materials
2.1 Isolation and Purification of GST-ETR1 Fusion Protein from Yeast
2.2 Histidine-Kinase Assay
3 Methods
3.1 Isolation and Purification of GST-ETR1 Fusion Protein from Yeast
3.2 Histidine Kinase Assay (See Note 12)
4 Notes
References
Chapter 9: Analysis of Ethylene Receptor Interactions by Co-immunoprecipitation Assays
1 Introduction
2 Materials
2.1 Growth of Plants in Liquid Culture
2.2 Membrane Protein Extraction
2.3 Co-IP Analysis
3 Methods
3.1 Growth of Plants in Liquid Culture
3.2 Membrane Protein Extraction
3.3 Co-IP Analysis
4 Notes
References
Chapter 10: Localization of the Ethylene-Receptor Signaling Complex to the Endoplasmic Reticulum: Analysis by Two-Phase Partitioning and Density-Gradient Centrifugation
1 Introduction
2 Materials
2.1 Growth of Plants in Liquid Culture
2.2 Membrane Isolation for Two-Phase Partitioning Analysis
2.3 Aqueous Two-Phase Partitioning Analysis of Arabidopsis Membranes
2.4 Membrane Isolation for Analysis of ER Association by Sucrose Density-Gradient Centrifugation
2.5 Sucrose Density-Gradient Centrifugation to Resolve ER by Density Shift
3 Methods
3.1 Growth of Plants in Liquid Culture
3.2 Membrane Isolation for Two-Phase Partitioning Analysis
3.3 Aqueous Two-Phase Partitioning Analysis of Arabidopsis Membranes
3.4 Membrane Isolation for Analysis of ER Association by Sucrose Density-Gradient Centrifugation
3.5 Sucrose Density-Gradient Centrifugation to Resolve ER by Density Shift
4 Notes
References
Chapter 11: Kinase Assay for CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1) in Arabidopsis thaliana
1 Introduction
2 Materials
3 Methods
3.1 In Vitro Kinase Assay Using EIN2 Protein as a Substrate
3.2 Measuring the Kinetics of CTR1 Kinase Using a Synthetic EIN2 Peptide (EIN2-PepS645A)
4 Notes
References
Chapter 12: Circular Dichroism and Fluorescence Spectroscopy to Study Protein Structure and Protein–Protein Interactions in Ethylene Signaling
1 Introduction
2 Materials
2.1 Preparation of ETR1
2.2 Preparation of EIN2
2.3 Instrumentation
3 Methods
3.1 CD Measurements
3.1.1 Secondary Structure Analysis of Ethylene Pathway Proteins
3.1.2 Conformational Changes of Ethylene Pathway Proteins upon Ligand-Binding
3.2 Tryptophan Fluorescence Measurements
4 Notes
References
Part III: Analysis of Ethylene Responses
Chapter 13: The Triple Response Assay and Its Use to Characterize Ethylene Mutants in Arabidopsis
1 Introduction
2 Materials
2.1 Preparation of Growth Media
2.2 Seed Sterilization
2.3 Top Agarose
2.4 Ethylene Treatment
2.4.1 Ethylene Treatment Setup
2.4.2 ACC Treatment Setup
2.5 Seeds
2.6 Phenotype Scoring
3 Methods
3.1 Media Preparation
3.2 Seed Sterilization
3.3 Plating
3.4 Seed Germination
3.5 Ethylene/ACC Treatment
3.6 Other Ethylene-Related Compounds to Augment the Triple Response Studies
3.7 Extension of the Method to Other Species
4 Notes
References
Chapter 14: Time-Lapse Imaging to Examine the Growth Kinetics of Arabidopsis Seedlings in Response to Ethylene
1 Introduction
2 Materials
2.1 Seed Preparation
2.2 Time-Lapse Imaging
2.3 Image Analysis
3 Methods
3.1 Seed Preparation
3.2 Time-Lapse Imaging
3.3 Analyzing Images
4 Notes
References
Chapter 15: Inhibitors of Ethylene Biosynthesis and Signaling
1 Introduction
2 Materials
2.1 Inhibitors
2.2 Seed and Plant Growth Media Preparation
2.3 Ethylene and 1-MCP Treatments
3 Methods
3.1 Preparation of Agar Plates
3.2 Seed Preparation and Plating (See Note 10)
3.3 Seedling Growth in the Presence of Soluble Inhibitors
3.4 Seedling Growth in the Presence of Gaseous Inhibitors
4 Notes
References
Chapter 16: Analysis of Growth and Molecular Responses to Ethylene in Etiolated Rice Seedlings
1 Introduction
2 Materials
3 Methods
3.1 Ethylene Treatment for Analysis of Seedling-Growth Response
3.1.1 Seed Germination
3.1.2 Ethylene Treatment
3.1.3 Phenotypic Investigation
3.2 Ethylene Treatment for Analysis of Gene Expression
3.3 1-MCP Treatment
4 Notes
References
Chapter 17: Love Me Not Meter: A Sensor Device for Detecting Petal Detachment Forces in Arabidopsis thaliana
1 Introduction
2 Materials
2.1 Breakstrength Meter Components
2.2 Plant Materials
3 Methods
3.1 Growth of Plant Materials
3.2 Calibration of the Breakstrength Meter
3.3 Preparing Plant for Measurement of Breakstrength (See Note 11)
3.4 Clamping of Petals
3.5 Measuring the Breakstrength
3.6 Recalibration
4 Notes
References
Chapter 18: Effects of Ethylene on Seed Germination of Halophyte Plants Under Salt Stress
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 19: Assessing Attraction of Nematodes to Host Roots Using Pluronic Gel Medium