Plant Iron Homeostasis: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: High-Throughput Plant Gene Expression Analysis by 384-Format Reverse Transcription-Quantitative PCR for Investigati...
1 Introduction
2 Materials
2.1 General Materials Required for Setting Up a Real-Time RT-qPCR Experiment (See Note 1)
2.2 PCR Reaction Setup for Mass Standard Preparation and qPCR Primer Verification
2.3 Sample Preparation and RT-qPCR
3 Methods
3.1 Design and Optimization of qPCR Oligonucleotide Primers
3.2 Mass Standard Preparation
3.3 Evaluation of RT-qPCR Primers
3.4 Plant Material Generation and Harvesting
3.5 RNA Preparation and cDNA Synthesis
3.6 qPCR Setup
3.7 Qualitative Analysis of qPCR Run and Gene Expression Data Visualization
4 Notes
References
Chapter 2: The Use of Spectral Imaging to Follow the Iron and pH-Dependent Accumulation of Fluorescent Coumarins
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Plant Growth Medium and Conditions
2.3 Reagents
2.4 Labware and Equipment
3 Methods
3.1 Plant Growth and Treatment
3.2 Spectral Library Setup
3.3 Coumarin Imaging
3.4 Linear Unmixing
4 Notes
References
Chapter 3: Assay of Fe(III) Chelate Reductase Activity in Arabidopsis thaliana Root
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Arabidopsis Plants
3.2 Ferric Reductase Assay
3.3 Result Presentation
4 Notes
References
Chapter 4: An Adapted Protocol for Quantitative Rhizosphere Acidification Assay
1 Introduction
2 Materials
2.1 Plant Materials and Media
2.1.1 Arabidopsis thalianaWildtype Seeds
2.1.2 Murashige and Skoog (MS) Medium Without Sucrose (for 500 mL)
2.1.3 Media With or Without Iron (See Note 2)
2.2 Solutions and Reagents
2.3 Equipment
3 Methods
3.1 Preparation of Plant Samples
3.2 Preparation of Assay Solution
3.3 Preparation of pH Standard Solutions
3.4 Incubation of Samples in Assay Solution
3.5 Generation of a Standard Curve
3.6 Measurement of Absorbance and Root Fresh Mass
3.7 Normalization of Sample Data
4 Notes
References
Chapter 5: Techniques to Study Common Root Responses to Beneficial Microbes and Iron Deficiency
1 Introduction
2 Materials
2.1 Equipment
2.2 Buffers, Media, and Solutions
2.2.1 Plant Cultivation
2.2.2 Preparing the WCS417 Inoculum
2.2.3 Harvesting Plant Material for Metabolomics or Gene Expression
2.2.4 GUS Staining
2.2.5 RNA Extraction and cDNA Synthesis
2.2.6 Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
3 Methods
3.1 Seed Sterilization and Sowing
3.2 Transplanting
3.3 Preparing the Bacterial Inoculum and Plant Inoculation
3.4 Harvesting Plant Material for qPCR and Coumarin Metabolite Profiling
3.5 GUS-Assisted Localization of Gene Expression
3.6 Coumarin Fluorescence Visualization
3.7 Coumarin Fluorescence Quantification
3.8 RNA Extraction and DNase Treatment
3.9 cDNA Synthesis
3.10 Quantitative Reverse-Transcription Polymerase Chain Reaction (qRT-PCR)
4 Notes
References
Chapter 6: Imaging and Quantifying the Endocytosis of IRON-REGULATED TRANSPORTER1 from Arabidopsis
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Medium Preparation
2.3 Additional Materials
2.4 Lab Equipment
3 Methods
3.1 Sample Preparation
3.2 Non-iron Metals Treatment and IRT1 Subcellular Localization
3.3 Dark Treatment and IRT1 Subcellular Localization
4 Notes
References
Chapter 7: Label-Free Quantitative Proteomics in Plant
1 Introduction
2 Materials
2.1 Protein Sample Preparation and Protein Concentration Determination
2.2 Protein Digestion
2.3 Peptide Desalt
2.4 MS/MS Analysis
2.5 Protein Identification and Quantification
3 Methods
3.1 Protein Sample Preparation
3.2 Protein Digestion and Peptide Desalt
3.3 LC-MS/MS Analysis
3.4 Protein Identification and Qualification
3.5 Bioinformatics
4 Notes
References
Chapter 8: Chromatin Immunoprecipitation (ChIP) to Study the Transcriptional Regulatory Network that Controls Iron Homeostasis...
1 Introduction
2 Materials
2.1 Plant Materials and Media
2.2 Other Reagents
2.3 Solutions
2.4 Labware and Equipment
3 Methods
3.1 Plant Growth and Treatment
3.2 Seedlings Collection and Fixation
3.3 Isolation of Nuclei and Shearing of Chromatin
3.4 Pre-clearing
3.5 ChIP and Reverse Crosslink
3.6 DNA Purification
3.7 Quantitative PCR and Data nalysis
4 Notes
References
Chapter 9: Comprehensive Survey of ChIP-Seq Datasets to Identify Candidate Iron Homeostasis Genes Regulated by Chromatin Modif...
1 Introduction
2 Comparative Survey of ChIP-Seq Datasets between Wild Type and Epigenetic Mutants
3 H3K4me2 and H3K4me3 Depositions via ATX3, ATX4, and ATX5
4 H3K27me3 Deposition via CLF or SWN
5 H3K36me3 Deposition via SDG8
6 H3ac Deposition via HAC1 and HAC5
7 H3Th3ph Deposition via AEL3 and AEL4
8 H2A.Z Substitution of Nucleosome via PIE1
9 Discussion
References
Chapter 10: Arabidopsis Micro-grafting to Study the Systemic Signaling of Nutrient Status
1 Introduction
2 Materials
2.1 Seed Sterilization
2.2 Plant Growth Medium
2.3 Grafting
3 Methods
3.1 Seed Sterilization
3.2 Preparing Agar Plates
3.3 Plant Growth
4 Notes
References
Chapter 11: Advances in Iron Retrograde Signaling Mechanisms and Uptake Regulation in Photosynthetic Organisms
1 Introduction
2 Systemic Plant Response to Fe Deficiency
3 Regulation of the Response to Fe Deficiency
4 Iron and Retrograde Signaling
5 PAP-SAL1 Pathway
6 Tetrapyrroles and Retrograde Signaling
7 Fe-S Cluster Biogenesis and Retrograde Signaling
References
Chapter 12: Functional Analysis of Chloroplast Iron Uptake and Homeostasis
1 Introduction
2 Materials
2.1 Plants and Materials for Plant Growth
2.2 Chloroplast Isolation and Purification
2.3 Determining Chloroplast Suspension Purity and Intactness
2.4 Iron Content Determination
2.5 Sample Preparation for Element Analysis by ICP-MS
2.6 Mössbauer Spectroscopy
2.7 Chloroplast Envelope Isolation
2.8 Ferric Chelate Reductase Activity
2.8.1 Alternative Method to Measure Ferric Chelate Reductase Activity
2.9 Transient Expression System to Prove Chloroplast Localization of Iron Homeostasis Elements
2.10 Protoplast Isolation
2.11 Protoplast Immunofluorescence Staining
3 Methods
3.1 Chloroplast Isolation and Purification
3.2 Isolation of Chloroplast Envelope Membranes
3.3 Determination of Intactness and Purity of the Chloroplast and the Purity of the Envelope Fractions by Western Blotting
3.4 Sample Preparation for Element Content Measurement by ICP-MS
3.5 Iron Species Determination by Mössbauer Spectroscopy
3.6 Iron Uptake Measurement
3.7 Chloroplast Envelope Ferric Chelate Reductase Activity Measurement
3.7.1 Ferric Chelate Reductase Activity Measurement on Solubilized Chloroplast Inner Envelope Samples (See Note 38)
3.8 Transient Expression to Test Chloroplast Targeting of Iron Homeostasis Elements
3.9 Protoplast Isolation
3.10 Localization of the Transiently Expressed GFP Fused Protein
4 Notes
References
Chapter 13: Perls/DAB Staining to Examine Iron Distribution in Arabidopsis Embryos
1 Introduction
2 Materials
2.1 Seed Dissection/Embryo Isolation
2.2 Perls Staining
2.3 DAB Intensification
3 Methods
3.1 Seed Dissection/Embryo Isolation
3.2 Perls Staining
3.3 DAB Intensification
4 Notes
References
Chapter 14: Visualizing Metal Distribution in Plants Using Synchrotron X-Ray Fluorescence Microscopy Techniques
1 Introduction
2 Materials
2.1 Mounting Materials
2.2 Sample Fixation and Embedding
2.3 Conventional 2D Scanning SXRF Equipment
2.4 Confocal SXRF Equipment
3 Methods
3.1 Sample Preparation and Mounting
3.1.1 Fresh Samples
Samples Mounted in a Wet Chamber
Live Plant Tissues
3.1.2 Seeds and Grains
3.1.3 Fixed Sample Preparation
3.2 Scanning
3.2.1 Conventional 2D SXRF Scanning
Optimizing for Low Concentrations
Example
Quantification
3.2.2 Confocal/3D Scanning SXRF
4 Notes
References
Chapter 15: A Simple Semi-hydroponic System for Studying Iron Homeostasis in Maize
1 Introduction
2 Materials
2.1 Stock Solutions
2.2 Nutrient Solution
2.3 Semi-hydroponic Equipment
2.3.1 Small-Scale System Equipment
2.3.2 Large-Scale System Equipment
3 Methods
3.1 Disinfecting and Setting Up the Semi-hydroponic System
3.2 Sterilizing and Germinating Maize Seeds
3.3 Preparing and Replenishing the Nutrient Solution
3.4 Shifting Plants to Different Iron Conditions
3.5 Collecting Tissues and Performing Gene Expression Measurements
4 Notes
References
Chapter 16: Optimizing Fe Nutrition for Algal Growth
1 Introduction
2 Materials
2.1 Acid Washing of Glass- and Plasticware
2.2 Prepare Trace Metal Grade Stock Solutions
2.3 Prepare TAP Growth Medium
3 Methods
3.1 Generate Starter Cultures
3.2 Acid Washing of Glass- and Plasticware
3.3 Experimental Growth of Chlamydomonas Cells in all Four Fe States
4 Notes
References
Index