Reactive Oxygen Species in Plants: Methods and Protocols (Methods in Molecular Biology, 2526)

✍ Scribed by Amna Mhamdi (editor)

• Publisher: Humana
• Year: 2022
• Tongue: English
• Leaves: 296
• Category: Library

✦ Synopsis

This detailed volume explores techniques to study reactive oxygen species (ROS) in plants and to characterize their roles in development and stress responses. Beginning with a section on strategies to induce ROS production, the book continues with methods to visualize ROS and detect changes in redox homeostasis, small-scale and targeted analyses for investigating the effects of ROS accumulation during stress on plant physiology and metabolism, as well as systems biology approaches to understand ROS functions. 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, Reactive Oxygen Species in Plants: Methods and Protocols serves as a vital resource that any researcher, and in particular young researchers, can use and adapt to further our knowledge of this dynamic area of plant science.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Strategies to Induce ROS Production
Chapter 1: Modification of Chloroplast Antioxidant Capacity by Plastid Transformation
1 Introduction
2 Materials
2.1 Preparation of Plant Materials
2.2 Culture Media Components
2.3 Vector for Tobacco Plastid Transformation
2.4 DNA-Coated Gold Particles and Biolistic Delivery System
2.5 Identification of Transplastomic Lines
2.6 GR Enzyme Activity Measurement
3 Methods
3.1 Preparation of Tobacco Plant Materials
3.2 Construction of Tobacco Plastid Transformation Vector
3.3 Particle Preparation and DNA Coating
3.4 Bombardment Procedure
3.5 Identification of Transplastomic Lines
3.6 GR Enzyme Activity Assay
4 Notes
References
Chapter 2: Analysis of Ascorbate Metabolism in Arabidopsis Under High-Light Stress
1 Introduction
2 Materials
3 Methods
3.1 High-Light Exposure and Sample Collection
3.2 Ascorbate Measurement
3.3 APX Activity
3.4 DHAR Activity
3.5 MDAR Activity
4 Notes
References
Chapter 3: Genetic Manipulation of Reactive Oxygen Species (ROS) Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice
1 Introduction
2 Materials
2.1 Generation of Gene-Edited Rice with Callus Method (See Note 1)
2.2 Cas9/Target-sgRNA Vector Construction
2.3 Agrobacterium Transformation and Infection of Callus
2.4 Validation of Targeted Mutations in Transgenic Plants
3 Methods
3.1 Induction of Calli
3.2 Vector Construction
3.3 Agrobacterium Transformation
3.4 Infection of Callus with Agrobacterium and Co-cultivation
3.5 Selection and Regeneration of Transgenic Plants
3.6 Cultivation of Transgenic Rice Plants
3.7 Evaluation of Targeted Mutations in Transgenic Rice Plants
4 Notes
References
Chapter 4: Studying Plant Stress Reactions In Vivo by PAM Chlorophyll Fluorescence Imaging
1 Introduction
1.1 Light Reactions of Photosynthesis
1.2 Basics of Chlorophyll Fluorometry
1.3 ETCs and Reactive Oxygen Species
1.4 Pharmacological Enhancement of ROS Signalling
2 Materials
2.1 Plant Growth
2.2 Chemical, Control Solutions, and Equipment
3 Methods
3.1 Working with Arabidopsis Seedlings on Horizontal Agar Plates
3.2 Working with Arabidopsis Rosettes Grown on Soil
3.3 Chlorophyll Fluorescence Imaging Using the IMAGING-PAM
3.4 Chlorophyll Fluorescence Imaging Using the FluorCam
4 Notes
References
Part II: Methods to Visualize ROS and to Detect Changes in Redox Homeostasis
Chapter 5: Live Monitoring of ROS-Induced Cytosolic Redox Changes with roGFP2-Based Sensors in Plants
1 Introduction
2 Materials
2.1 Plant Material and Growth Conditions
2.2 Assay Media and Stock Solutions
2.3 Confocal Laser Scanning Microscopy and Perfusion Setup
2.4 Plate Reader Setup
2.5 Image and Data Analysis Software
3 Methods
3.1 CLSM Methods
3.1.1 Sample Mounting for Steady-State Measurements
3.1.2 Perfusion System Setup
3.1.3 Elicitor-Induced ROS Generation and Sensor Calibration
3.1.4 CLSM Settings
3.1.5 Data Collection and Analysis
3.2 Plate Reader Methods
3.2.1 Sample Mounting of Whole Seedlings
3.2.2 Sample Mounting of Leaf Discs
3.2.3 Settings for Spectral Measurements
3.2.4 Settings for Ratiometric Time Course Measurements
3.2.5 Elicitor-Induced ROS Generation
3.2.6 Data Collection and Analysis
4 Notes
References
Chapter 6: Quantitative Measurement of Ascorbate and Glutathione by Spectrophotometry
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Equipment
3 Methods
3.1 Extraction
3.2 Neutralization
3.3 Treatment of Extract Aliquots to Distinguish Between Oxidized and Reduced Forms
3.3.1 Preparation of the GSSG Assay
3.3.2 Reduction of Dehydroascorbate (DHA) for Assay of Total Ascorbate
3.4 Assays
3.4.1 Ascorbate
3.4.2 Total Glutathione
3.4.3 Total Ascorbate
3.4.4 GSSG
3.5 Data Processing
4 Notes
References
Chapter 7: Measurement of NAD(P)H and NADPH-Generating Enzymes
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Equipment
3 Methods
3.1 Quantification of Pyridine Nucleotides
3.1.1 Metabolite Extraction and Neutralization
3.1.2 Quantification of NAD+ and NADH
3.1.3 Quantification of NADP and NADPH
3.2 Assays of NADPH-Generating Enzymes
3.2.1 Measurement of NADP+-Dependent Isocitrate Dehydrogenase
3.2.2 Measurement of Glucose-6-Phosphate Dehydrogenase
4 Notes
References
Chapter 8: Quantitative Analysis for ROS-Producing Activity and Regulation of Plant NADPH Oxidases in HEK293T Cells
1 Introduction
2 Materials
2.1 Media and Buffers
2.2 Solutions
2.3 Equipment
3 Methods
3.1 Plasmid DNA Construction and Purification
3.2 Subculture of HEK293T Cell and Preparation of 96 Well Plate for ROS Assay
3.3 Transfection
3.4 ROS Measurement
3.5 Representative Data
3.6 Protein Extraction for Western Blot Analysis
3.7 Starting Cell Culture from Frozen HEK293T Cells Stock
3.8 Freezing Cells for New Frozen Stocks
4 Notes
References
Part III: Small-Scale Targeted Analysis of ROS Accumulation During Stress and Effects on Plant Physiology
Chapter 9: Estimation of the Level of Abasic Sites in Plant mRNA Using Aldehyde Reactive Probe
Abbreviations
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Equipment
3 Methods
3.1 Plant Cultivation and Treatment
3.2 Isolation of Total RNA
3.3 Binding of ARP with mRNA Abasic Sites
3.4 Detection and Quantification of Abasic Sites in mRNA
4 Notes
References
Chapter 10: A Simplified Method to Assay Protein Carbonylation by Spectrophotometry
1 Introduction
2 Materials
3 Methods
3.1 Sample Extraction and Clarification
3.2 Removal of Nucleic Acids
3.3 Derivatization with DNPH
3.4 Removal of Unincorporated DNPH by Gel Filtration
3.5 Quantification of Carbonyls
4 Notes
References
Chapter 11: In Vitro Biochemical Analysis of Recombinant Plant Proteins Under Oxidation
1 Introduction
2 Materials
2.1 E. coli Cell Line and Plasmids
2.2 Reagents and Buffers
2.2.1 Reagents
2.2.2 Buffers
2.2.3 Equipments
3 Methods
3.1 Design Expression Constructs in Escherichia coli (E. coli ) Cell
3.1.1 Select Expression Vector
3.1.2 Select an Affinity Tag
3.1.3 Select an Expression Cell Line
3.1.4 Delete Transit Peptides
3.1.5 Optimize Codon Usage of Your DNA Sequence (Optional) (See Note 7)
3.2 Protein Expression
3.2.1 Test Small-Scale Protein Expression
3.2.2 Prepare Protein Samples for Small-Scale Expression Analysis
3.2.3 SDS-PAGE Gel and Western Blot Analysis for Protein Expression and Solubility
3.2.4 Upscale Protein Expression
3.3 Protein Purification
3.3.1 On-bench Ni-IMAC
3.3.2 Ni-IMAC with ÄKTA
3.3.3 On-bench IEC
3.3.4 IEC with ÄKTA
3.3.5 On-bench Desalting
3.3.6 SEC with ÄKTA
3.4 Reducing and Oxidizing Proteins
3.5 Non-reducing and Reducing SDS-PAGE
3.6 Detection of Protein S-Sulfenylation by Dimedone
4 Notes
References
Chapter 12: Methods to Analyze the Redox Reactivity of Plant Proteins
1 Introduction
2 Materials
2.1 Solutions and Reagents
2.2 Equipment
3 Methods
3.1 Determine the Impact of the Redox Environment on Proteins Oligomerization
3.1.1 Preparation of Purified Recombinant Protein Samples
3.1.2 Treatment of Target Proteins with Reducing and Oxidizing Agents
3.1.3 Separation and Detection of Monomeric and Multimeric Protein Products Using SEC-HPLC
3.2 Determine the Impact of the Redox Modifications on Enzyme Activity
3.2.1 Redox Treatment of Purified Recombinant Protein Preparations
3.2.2 Analysis of Total Protein Extracts Obtained from Plants Subjected to Oxidative Stress
3.2.3 Detection of Peptidase Activity by Fluorometric Assay
3.2.4 Enzyme Activity Analysis
3.2.5 Redox Titration Analysis
4 Notes
References
Chapter 13: Determination of ROS-Induced Lipid Peroxidation by HPLC-Based Quantification of Hydroxy Polyunsaturated Fatty Acids
1 Introduction
2 Materials
2.1 Biological Material
2.2 Equipment for the Extraction of Hydroxy Fatty Acids
2.3 Solutions for the Extraction of Hydroxy Fatty Acids
2.4 HPLC Analysis
3 Methods
3.1 Total Lipid Extraction
3.2 Evaporation and Saponification
3.3 Quantification Hydroxy Fatty Acid by HPLC
3.4 Calculation of Enzymatic and Non-enzymatic Lipid Peroxidation
3.5 A Variant of the Method
4 Notes
References
Chapter 14: Detection of Lipid Peroxidation-Derived Free Azelaic Acid, a Biotic Stress Marker and Other Dicarboxylic Acids in ...
1 Introduction
2 Materials
2.1 Materials for Preparation of Leaf Tissue Samples
2.2 Materials for Preparation of Petiolar Exudates
2.3 Reversed-Phase HPLC-MS Analysis
3 Methods
3.1 Leaf Tissue
3.2 Petiolar Exudates
3.3 HPLC-MS Analysis
4 Notes
References
Chapter 15: Determination of Reactive Carbonyl Species, Which Mediate Reactive Oxygen Species Signals in Plant Cells
1 Introduction
1.1 Reactive Carbonyl Species (RCS) Mediate ROS Signaling
1.2 Comprehensive Analysis of Carbonyls Providing Key Information to the RCS Functions
2 Materials
2.1 Plant Growth and Treatment
2.2 Preparation of DNP-Carbonyl Standards
2.3 Extraction and Derivatization of Carbonyls
2.4 HPLC Analysis
3 Methods
3.1 Recrystallization of DNPH
3.2 Preparation of Plant Materials
3.3 Treatment of Tobacco BY-2 Cells with H2O2
3.4 Treatment of Tobacco Leaf Epidermis with ABA and MeJA
3.5 Treatment of Arabidopsis and Tobacco Roots with Auxin and H2O2
3.6 Extraction and DNP-Derivatization of Carbonyls in Plant Tissues
3.7 HPLC Analysis and Data Processing
3.8 Determination of the Conversion Factor k
4 Notes
References
Chapter 16: Measuring Stress-Induced Changes in Defense Phytohormones and Related Compounds
1 Introduction
2 Materials
2.1 Reagents and Solvents
2.2 Equipment and Software
3 Methods
3.1 Growth Conditions and Sampling
3.2 Extraction
3.2.1 Methanol Extraction and SPE
3.2.2 Acetone Extraction
3.3 Sample Preparation and Liquid Chromatography
3.4 Mass Spectrometry
3.5 Data Analysis and Processing
3.5.1 Identification
3.5.2 Quantification
4 Notes
References
Part IV: Systems Biology Approaches to Understand ROS Functions
Chapter 17: Targeted Mass Spectrometry Analysis of Protein Phosphorylation by Selected Ion Monitoring Coupled to Parallel Reac...
1 Introduction
2 Materials
2.1 Solutions (See Note 1)
2.2 Equipment
2.3 Software
3 Methods
3.1 Generation of a Spectral Library and an Isolation List
3.2 Isolation of Protein Samples
3.3 Sample Preparation by In-Gel Protein Digestion for Mass Spectrometry Analysis
3.3.1 In-Gel Sample Denaturation and Purification (See Note 9)
3.3.2 Protein Reduction and Alkylation
3.3.3 Protein Digestion, Peptide Extraction, and Vacuum Drying
3.4 Mass Spectrometry Analysis
3.4.1 Finalization of Sample Preparation for Mass Spectrometry
3.4.2 Running tSIM/PRM
3.5 Data Analysis
4 Notes
References
Chapter 18: Quantitative Analysis of Posttranslational Modifications of Plant Histones
1 Introduction
2 Materials
2.1 Plant Material
2.2 Nuclei Enrichment and Histone Extraction
2.3 Chemical Derivatization of Histones
2.4 Sample Preparation for Mass Spectrometry
2.5 LC-MS/MS
2.6 Data Processing
3 Methods
3.1 Nuclei Isolation and Histone Extraction
3.2 Chemical Derivatization of Lysines (Protein Level)
3.3 Protein Purification and Enzymatic Digestion
3.4 Chemical Derivatization of Peptide N-Termini (Peptide Level)
3.5 Desalting and Sample Preparation for LC-MS/MS
3.6 LC-MS/MS
3.7 Data Analysis
3.7.1 Peptide Identification
3.7.2 Peptide Form Quantification
4 Notes
References
Chapter 19: Characterization of RBPome in Oxidative Stress Conditions
1 Introduction
2 Materials
2.1 Plant Materials, Reagents and Solutions
2.2 Equipments
3 Methods
3.1 Incubation of Suspension Cell Culture with H2O2
3.2 In Vivo Crosslinking of mRNPs and Harvesting of Cells
3.3 Preparation of Oligo-d(T)25 Magnetic Bead Suspension
3.4 Lysis of PSB-D Cells and Homogenizing of Cell Lysate
3.5 Pull-down and Purification of Crosslinked mRNPs by Oligo-d(T)25 Beads
3.6 Reuse and Regeneration of Oligo-d(T)25 Beads
3.7 Quantity and Quality Control of the Samples
3.7.1 Captured RNAs
3.7.2 Captured RBPs
3.8 Sample Preparation for LC-MS/MS Analysis
3.9 LC-MS/MS Analysis
3.10 OxRBPome Identification
4 Notes
References
Chapter 20: Analysis of ROS-Triggered Changes in the Transcriptome
1 Introduction
2 Materials
3 Methods
3.1 Experimental Design, Library Construction, and Sequencing
3.2 Preprocessing and Genome Alignment
3.3 Differential Expression Analysis Using edgeR
4 Notes
References
Index