Plant Mitochondria: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Purification of Leaf Mitochondria from Arabidopsis thaliana Using Percoll Density Gradients
	1 Introduction
	2 Materials
		2.1 Arabidopsis Growth in  Soil
		2.2 Arabidopsis Growth in Shaking Liquid Culture Flasks
		2.3 Purification of Mitochondria Using Continuous Percoll Density Gradients
	3 Methods
		3.1 Arabidopsis Growth in  Soil
		3.2 Arabidopsis Grown in Liquid Half-Strength MS Medium (Fig. 1)
		3.3 Preparation of Continuous Percoll Density Gradients
		3.4 Purification of Arabidopsis Mitochondria
	4 Notes
	References
Chapter 2: Cell Type-Specific Isolation of Mitochondria in Arabidopsis
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Compounds
		2.3 Buffers and Solutions
	3 Methods
		3.1 Promoter Cloning and Plant Transformation
		3.2 Plant Selection and Growth
		3.3 Crude Mitochondria Extraction
		3.4 Preparation of Magnetic Beads
		3.5 Isolation of Biotinylated Mitochondria
	4 Notes
	References
Chapter 3: Isolation of Plant Mitochondria Using Affinity Purification
	1 Introduction
	2 Materials
		2.1 Plant Cultivation
		2.2 Mitochondria Isolation
		2.3 Yield Estimation
		2.4 Quality Control
		2.5 Preparation of a 1 mM Cytochrome c Solution
	3 Methods
		3.1 Cultivation of Arabidopsis Seedlings
		3.2 Cultivation of Arabidopsis Rosette Leaves
		3.3 Cultivation of Arabidopsis Roots
		3.4 Isolation of Mitochondria Via Immunocapture with Magnetic Anti-HA Beads
		3.5 Estimation of Yield by Determination of Total Protein
		3.6 Estimation of Intactness
		3.7 Estimation of Recovery
	4 Notes
	References
Chapter 4: Isolation of Highly Purified, Intact, and Functional Mitochondria from Potato Tubers Using a Two-in-One Percoll Den...
	1 Introduction
	2 Materials
		2.1 Homogenization and Filtration
		2.2 Differential Centrifugation
		2.3 Percoll Gradient Centrifugation and Final  Wash
	3 Methods
		3.1 Tissue Disruption
		3.2 Differential Centrifugation
		3.3 Gradient Centrifugation
		3.4 Final Washing of Mitochondria
		3.5 Characterization of the Gradient Profile (Optional)
	4 Notes
	References
Chapter 5: Integrity Assessment of Isolated Plant Mitochondria
	1 Introduction
	2 Materials
		2.1 General Materials
		2.2 Complex I Latency Assay
		2.3 Spectrophotometric Coupled Assay
		2.4 CCO Latency Assay
	3 Methods
		3.1 Complex I Latency
		3.2 Spectrophotometric Coupled Assay (See Fig. 3)
		3.3 CCO Latency Assay
	4 Notes
	References
Chapter 6: High-Throughput Oxygen Consumption Measurements in Leaf Tissue Using Oxygen Sensitive Fluorophores
	1 Introduction
	2 Materials
		2.1 Gas Phase Leaf OCR Measurements
		2.2 Floating Leaf Disc OCR Measurements
	3 Methods
		3.1 Gas Phase Leaf OCR Measurements Using the Q2
		3.2 Gas Phase Leaf OCR Measurements Using the SDR SensorDish System
		3.3 Floating Leaf Disc OCR Measurements During Incubations in External Metabolite Solutions
		3.4 Data Analysis
		3.5 Experimental Design of OCR Screens
	4 Notes
	References
Chapter 7: Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin
	1 Introduction
	2 Materials
		2.1 General Materials
		2.2 In Situ Assay of Mitochondrial NAD-ICDH
		2.3 Assay of ETC Activity
	3 Methods
		3.1 In Situ Assay of Mitochondrial NAD-Isocitrate Dehydrogenase (ICDH) (See Note 10)
		3.2 Assay of ETC Activity (See Fig. 1 and Note 13)
	4 Notes
	References
Chapter 8: Assessing the Kinetics of Metabolite Uptake and Utilization by Isolated Mitochondria Using Selective Reaction Monit...
	1 Introduction
	2 Materials
		2.1 Mitochondrial Feeding
		2.2 Metabolite Extraction and Derivatization
		2.3 Metabolite Quantitation by SRM-MS
	3 Methods
		3.1 Mitochondrial Feeding
		3.2 Metabolite Extraction and Derivatization
			3.2.1 Metabolite Extraction
			3.2.2 Metabolite Derivatization
			3.2.3 Preparation and Derivatization of Individual and Pooled Metabolite Standards
		3.3 Metabolite Quantification by SRM-MS
			3.3.1 Optimization of Metabolite Transitions for SRM-MS
			3.3.2 Data Acquisition
			3.3.3 Data Analysis
	4 Notes
	References
Chapter 9: Complexome Profiling of Plant Mitochondrial Fractions
	1 Introduction
	2 Materials
		2.1 Components for Casting and Running a BN  Gel
		2.2 Components for Solubilization of Mitochondrial Proteins and Protein Complexes
		2.3 Components for Protein Analyses by Mass Spectrometry
	3 Methods
		3.1 BN Gel Preparation
		3.2 BN-PAGE Sample Preparation
		3.3 Running the BN  Gel
		3.4 Fractionation of BN Gel Lanes and in Gel Tryptic Digestion
		3.5 Liquid Chromatography Coupled Tandem Mass Spectrometry (LC-MS/MS)
		3.6 Database Search and Heat Map Building
	4 Notes
	References
Chapter 10: High-Throughput BN-PAGE for Mitochondrial Respiratory Complexes
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 BN-PAGE
		2.3 Immunoblotting
		2.4 Complex I Activity Staining
	3 Methods
		3.1 Total Membrane Extraction from Leaves
		3.2 BN-PAGE
		3.3 Transfer
		3.4 Immunodetection
		3.5 Complex I Activity Staining
	4 Notes
	References
Chapter 11: Assessment of Mitochondrial Protein Composition and Purity by Mass Spectroscopy
	1 Introduction
	2 Materials
		2.1 Gel Fractionation, Concentration, and Purification for the Identification of Proteins to Determine Mitochondrial Purity
			2.1.1 Mitochondrial Precipitation and Resuspension
			2.1.2 SDS-PAGE of Mitochondrial Proteins
			2.1.3 Gel Staining
			2.1.4 Extraction and Digestion of Proteins from 1D-SDS-PAGE
			2.1.5 Identification of Proteins from SDS-PAGE  Gels
		2.2 Gel-Free Peptide Fractionation and Identification to Determine Mitochondrial Purity
			2.2.1 Sample Preparation
			2.2.2 Sample Fractionation and Mass Spectrometry
		2.3 SRM Mass Spectrometry to Determine Mitochondrial Purity
			2.3.1 Selection and Optimization of SRM Transitions
			2.3.2 Determining the Abundance of Target Proteins
	3 Methods
		3.1 Gel Fractionation, Concentration, and Purification for the Identification of Proteins to Determine Mitochondrial Purity
			3.1.1 Mitochondrial Precipitation and Resuspension
			3.1.2 Mitochondrial Fractionation and Staining
			3.1.3 Mitochondrial Concentration
			3.1.4 Extraction and Digestion of Proteins for Identification
			3.1.5 Identification of Proteins
		3.2 Gel-Free Peptide Fractionation and Identification to Determine Mitochondrial Purity
			3.2.1 Sample Preparation
			3.2.2 Sample Fractionation and Mass Spectrometry
			3.2.3 Data Analysis
		3.3 SRM Mass Spectrometry to Determine Mitochondrial Purity
			3.3.1 Selection and Optimization of SRM Transitions
			3.3.2 Determining the Abundance of Target Proteins
	4 Notes
	References
Chapter 12: Mitochondrial GFP-Tagged Protein Localization Using Transient Transformations in Arabidopsis thaliana
	1 Introduction
	2 Materials
		2.1 Plant Lines and Growth Conditions
		2.2 Transient Transformation of Arabidopsis Protoplasts
		2.3 Agroinfiltration of Arabidopsis Leaves
		2.4 Confocal Microscope Imaging
	3 Methods
		3.1 Growth of Plants
		3.2 Mitochondrial GFP-Tagged Protein Localization in Arabidopsis thaliana Protoplasts
		3.3 Mitochondrial GFP-Tagged Protein Localization by Agroinfiltration of Arabidopsis thaliana Leaves
		3.4 Confocal Microscope Imaging
	4 Notes
	References
Chapter 13: Assessment of Mitochondrial Protein Topology and Membrane Insertion
	1 Introduction
	2 Materials
		2.1 Carbonate Extraction
		2.2 Generation of Mitoplasts
		2.3 Solubilization of Membrane Proteins
		2.4 Proteinase K Digestion
		2.5 TCA Precipitation
		2.6 In Vivo Split-GFP Approach for Subcellular Localization of Proteins: Cloning Strategy
		2.7 Transient Protein Expression in Nicotiana benthamiana
		2.8 Protoplast Isolation and Visualization of Self-Assembling GFP
	3 Methods
		3.1 Membrane Integration of Mitochondrial Proteins
		3.2 Topology of Mitochondrial Membrane Proteins in Mitochondria and Mitoplasts
		3.3 Topology of Mitochondrial Membrane Proteins in Solubilized Membranes
		3.4 Generation of Self-Assembling GFP Constructs by Golden Gate Cloning
		3.5 Transient Expression in Tobacco Leaves Mediated by Agrobacterium Transformation
		3.6 Protoplast Isolation
		3.7 Subcellular Localization of Mitochondrial Membrane Proteins Demonstrated by In Vivo Labeling with Self-Assembling GFP
	4 Notes
	References
Chapter 14: Assessment of Protein Synthesis in Mitochondria Isolated from Rosette Leaves and Liquid Culture Seedlings of Arabi...
	1 Introduction
	2 Materials
		2.1 Preparation of Plant Material
		2.2 Labeling of Proteins in Isolated Mitochondria
		2.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Coomassie Gel Staining
		2.4 Drying of Gels and Visualization of Radiolabeled Proteins by Autoradiography
	3 Methods
		3.1 Preparation of Plant Material
		3.2 Isolation of Mitochondria
		3.3 Labeling of Proteins in Isolated Mitochondria
		3.4 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Coomassie Gel Staining
		3.5 Drying of Gels and Visualization of Radiolabeled Proteins by Autoradiography
	4 Notes
	References
Chapter 15: Markers for Mitochondrial ROS Status
	1 Introduction
	2 Materials
		2.1 Estimation of Superoxide Formation
			2.1.1 Equipment
			2.1.2 Reagents and Solutions
		2.2 Lipid Peroxidation Assay
			2.2.1 Equipment
			2.2.2 Reagents and Solutions
		2.3 Detection of Carbonylated Proteins
			2.3.1 Equipment
			2.3.2 Reagents and Solutions
		2.4 Abundance and Redox State of Glutathione
			2.4.1 Equipment
			2.4.2 Reagents and Solutions
	3 Methods
		3.1 Estimation of Superoxide Formation
		3.2 Lipid Peroxidation Assay
		3.3 Detection of Carbonylated Proteins
		3.4 Abundance and Redox State of Glutathione
	4 Notes
	References
Chapter 16: Mass Spectrometry-Based Quantitative Cysteine Redox Proteome Profiling of Isolated Mitochondria Using Differential...
	1 Introduction
	2 Materials
		2.1 Protein Extraction and Labeling of Free  Cys
		2.2 Protein Digestion
		2.3 Desalting of Peptides onC18 Stop-and-Go-Extraction Tips (Stage Tips)
		2.4 Affinity Enrichment Using Anti-TMT Antibody
		2.5 LC-MS/MS Measurement and Data Evaluation
	3 Methods
		3.1 Protein Extraction and Labeling of Free  Cys
		3.2 Protein Digest
		3.3 Desalting of Peptides on C18 Stop-and-Go-Extraction Tips (Stage Tips)
		3.4 Label Distribution and Labeling Efficiency
		3.5 Immunoprecipitation of TMT-Containing Peptides
		3.6 Guidelines for LC-MS/MS Analysis
		3.7 Data Analysis
	4 Notes
	References
Chapter 17: Analysis of the Plant Mitochondrial Transcriptome
	1 Introduction
	2 Materials
		2.1 Total RNA Isolation
		2.2 Quantitative RT-PCR
		2.3 RNA-Seq
		2.4 RNA-Seq Analysis
		2.5 Northern Blotting
	3 Methods
		3.1 Total RNA Isolation
		3.2 Quantitative RT-PCR Based Mitochondrial Transcript Analysis Assay
		3.3 RNA-Seq Based Mitochondrial Transcript Analysis Assay
		3.4 Northern Blot Using Biotinylated Oligonucleotides
	4 Notes
	References
Chapter 18: Quantification of Mitochondrial RNA Editing Efficiency Using Sanger Sequencing Data
	1 Introduction
	2 Materials
		2.1 Software
		2.2 Kits and Enzymes
		2.3 Primers
	3 Methods
		3.1 Preparation of Total Cellular  RNA
		3.2 RT-PCR
		3.3 DNA Sequencing
		3.4 Preparation of a Reference File for Mapping Sequencing  Data
		3.5 Mapping of Sanger Sequencing Results
		3.6 Outputting C and T Signal Information of RNA Editing Sites
	4 Notes
	References
Chapter 19: Nascent Transcript Sequencing for the Mapping of Promoters in Arabidopsis thaliana Mitochondria
	1 Introduction
	2 Materials
		2.1 Growth of Arabidopsis Seedlings in Liquid Culture and Isolation of Mitochondria
			2.1.1 Equipment
			2.1.2 Materials
			2.1.3 Reagents and Solutions
		2.2 Nascent RNA Labeling by Transcriptional Run-on
			2.2.1 Equipment
			2.2.2 Materials, Reagents, and Solutions
		2.3 Enrichment of BrU-Labeled Nascent  RNA
			2.3.1 Equipment
			2.3.2 Materials, Reagents, and Solutions
		2.4 Depletion of Processed Transcripts and NGS Library Preparation
			2.4.1 Equipment
			2.4.2 Materials, Reagents, and Solutions
		2.5 Read Mapping and Identification of  TSS
			2.5.1 Resources
	3 Methods
		3.1 Growth of Arabidopsis Seedlings in Liquid Culture and Isolation of Mitochondria
		3.2 Run-on Transcription and Nascent Transcript Labeling
		3.3 Enrichment of BrU-Labeled Nascent  RNA
			3.3.1 Preparation of Anti-BrdU Agarose Beads
			3.3.2 Enrichment of BrU-Labeled Nascent RNA with Anti-BrdU Agarose Beads
		3.4 Depletion of Processed Transcripts and NGS Library Preparation
		3.5 Read Mapping and Identification of  TSS
	4 Notes
	References
Chapter 20: Assessment of Mitochondrial DNA Copy Number, Stability, and Repair in Arabidopsis
	1 Introduction
		1.1 Determination of mtDNA Copy Numbers
		1.2 Analysis of Recombination Activities Involving Small Repeated Sequences
		1.3 Relative Stoichiometry of the mtDNA Sequences
		1.4 Treatments and Effects of Ciprofloxacin
	2 Materials
		2.1 DNA Extraction from Arabidopsis Seedlings and Ciprofloxacin Treatment
		2.2 qPCR Reactions
	3 Methods
		3.1 DNA Extraction from Arabidopsis Seedlings Grown In Vitro
			3.1.1 Sterilization and Growth of Seedlings
			3.1.2 Total DNA Extraction by the CTAB Method
		3.2 Relative Quantification of mtDNA Copy Numbers by qPCR
		3.3 Analysis of Recombination Activities Involving Small Repeated Sequences
		3.4 Relative Stoichiometry of the mtDNA Sequences
		3.5 Ciprofloxacin Treatment
	4 Notes
	References
Chapter 21: Measuring the Activity of DNA Repair Enzymes in Isolated Mitochondria
	1 Introduction
	2 Materials
		2.1 Mitochondrial Fraction
		2.2 5′-End-Labeling and Annealing of Oligonucleotides
		2.3 Annealing of Oligonucleotides for DNA Polymerase and DNA Ligase Activity Assay
		2.4 BER Assays
			2.4.1 Lysis of Mitochondria
			2.4.2 DNA Glycosylase Activity Assay
			2.4.3 AP Endonuclease Activity Assay
			2.4.4 DNA Polymerase and DNA Ligase Activity
		2.5 Electrophoresis and Analysis of Resulting Image
	3 Methods
		3.1 Producing Labelled and Lesion-Containing Oligonucleotides for the Assays
			3.1.1 5′-End-Labeling of Lesion-Containing Oligonucleotides with [γ-32P] ATP and Annealing for Incision Assays
			3.1.2 Annealing of Lesion-Containing Oligonucleotides for DNA Polymerase and DNA Ligase Activity Assay
			3.1.3 Electrophoresis-Native Conditions
			3.1.4 Lysis of Mitochondria
		3.2 Base Excision Repair Assays
			3.2.1 DNA Glycosylase Assays
			3.2.2 AP Endonuclease Assay
			3.2.3 DNA Polymerase and DNA Ligase Activity Assay
		3.3 Electrophoresis-Denaturing Conditions
		3.4 Interpretation of the Results and Calculation of Enzymatic Activity
	4 Notes
	References
Chapter 22: MitoTALENs: A Method for Targeted Gene Disruption in Plant Mitochondrial Genomes
	1 Introduction
	2 Materials
		2.1 Plant Materials
		2.2 Cloning Materials
	3 Methods
		3.1 Selection of Target Sequences
		3.2 Vector Construction
		3.3 Transformation and Selection of Transformants
	4 Notes
	References
Index