Catharanthus roseus: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: RNA In Situ Hybridization of Paraffin Sections to Characterize the Multicellular Compartmentation of Plant Secondar...
1 Introduction
2 Materials
2.1 Riboprobe In Vitro Transcription
2.1.1 Vectors
2.1.2 Molecular Biology Reagents
2.1.3 Software and Equipment
2.2 Materials for Sample Preparation
2.3 Medium-Throughput In Situ Hybridization Conditions
2.3.1 RNase-Free Conditions and Treatment of Material
2.3.2 Equipment
2.3.3 Specific Materials
2.3.4 Solvents and Chemicals
2.4 Buffers
3 Methods
3.1 Digoxigenin-Labeled Riboprobe In Vitro Transcription
3.1.1 Plasmid Amplification in Bacteria
3.1.2 Plasmid Linearization
3.1.3 Digoxigenin-Labeled Riboprobe In Vitro Transcription (see Note 17)
3.1.4 Alkaline Hydrolysis of Digoxigenin-Labeled Riboprobes (see Note 23)
3.1.5 Quality and Quantification Control of Riboprobes (see Note 25)
3.2 Tissue Sampling and Embedding in Tissue Microarrays (see Note 29)
3.2.1 Fixation
3.2.2 Dehydration and Paraffin Infiltration
3.2.3 Embedding and Tissue Microarray Block Preparation
3.3 Medium-Throughput In Situ Hybridization Protocol (see Note 38)
3.3.1 Microtomy
3.3.2 Paraplast Plus Removal and Prehybridization
3.3.3 Hybridization
3.3.4 Stringent Washing
3.3.5 Immunological Detection of Hybridized Probes
3.3.6 Color Development of AP Reaction
3.3.7 Slide Permanent Mounting and Scanning
3.3.8 Result Analysis and Figure Mounting
3.4 Conclusion
4 Notes
References
Chapter 2: Imaging MS Analysis in Catharanthus roseus
1 Introduction
2 Material
2.1 Plant Material
2.2 LC-MS Measurement
2.3 Equipment for Imaging MS
2.4 Software
3 Methods
3.1 LC-MS Analysis of Plant Tissue
3.2 Preparation of Plant Tissues for Imaging MS
3.3 Imaging MS Measurement
3.4 Analysis of Metabolome Data
4 Notes
References
Chapter 3: Surface-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry (SALDI-IMS)-Based Detection of Vinca Alkaloi...
1 Introduction
2 Materials
2.1 Functionalized TiO2 Nanowire Plate Preparation
2.2 Petal Imprint Preparation
2.3 Instrumentation
3 Methods
3.1 Functionalized TiO2 Nanowire Plate Preparation
3.2 Petal Imprint Preparation
3.3 Mass Spectrometry Imaging
4 Notes
References
Chapter 4: Sample Preparation, Data Acquisition, and Data Analysis for 15N-Labeled and Nonlabeled Monoterpene Indole Alkaloids...
1 Introduction
2 Materials
2.1 Plant Culture
2.2 Labeling and Sample Preparation
2.3 Analysis and Data Processing
3 Methods
3.1 15N Labeling to Plants (See Note 1)
3.2 Sample Preparation
3.3 Untargeted Analysis (See Note 5)
3.4 Data Processing
3.5 Alkaloid Network Analysis (See Note 9)
3.6 Conclusion
4 Notes
References
Chapter 5: Studying Iridoid Transport in Catharanthus roseus by Grafting
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Plant Culture
2.3 In Vitro Manipulations
2.4 Media
2.5 Sample Analyses
3 Methods
3.1 Preparation of Plant Materials
3.1.1 Sterilizing C. roseus Seeds
3.1.2 Sterilizing C. roseus Cuttings
3.2 Grafting C. roseus Plants (See Note 1)
3.3 Feeding Iridoids to C. roseus Roots (See Note 6)
3.4 Sample Analyses from Grafts and Feeding Assays
4 Notes
References
Chapter 6: Pictet-Spengler Reaction for the Chemical Synthesis of Strictosidine
1 Introduction
2 Materials
2.1 Reagents and Solvents
2.2 Equipment
3 Methods
3.1 Pictet-Spengler Reaction
3.2 Collection of Strictosamide and Vincosamide
3.3 Collection of Strictosidine
3.4 Purification of Strictosidine by Flash Chromatography on Silica Gel
3.5 Analysis of Strictosidine by NMR
4 Notes
References
Chapter 7: Implementation of an MS/MS Spectral Library for Monoterpene Indole Alkaloids
1 Introduction
2 Materials
2.1 Solvents and Reagents
2.2 Equipment
2.2.1 Plant Alkaloid Fractionation
2.2.2 Sample Preparation
2.2.3 LC-MS/MS Data Acquisition (See Note 4)
2.2.4 Data Processing
3 Methods
3.1 Sample Preparation for LC-MS/MS Data Acquisition (See Notes 5 and 6)
3.2 LC-MS/MS Data Acquisition
3.2.1 UHPLC System Setup for Analyzing MIA
3.2.2 Q-TOF MS Parameters
3.3 Data Mining for the Implementation of MS/MS Spectral Library
3.3.1 MS/MS Spectra Mining and GNPS Upload Using MassHunter v10.0
3.3.2 MS/MS Spectra Mining and GNPS Upload Using MZmine 2.53
3.3.3 Data Mining and Batch Mode Upload to the GNPS Using MSMS-Chooser Workflow
3.4 MN-Annotation of MIA-Rich Plant Extract
4 Notes
References
Chapter 8: Semisynthesis of Bis-Indole Alkaloid (-)-Melodinine K Enabled by a Combination of Biotransformation and Chemical Sy...
1 Introduction
2 Materials
2.1 Culture Media and Yeast Stain
2.2 Chemicals
2.3 Materials and Apparatus
3 Methods
3.1 Bioproduction of (-)-16-Hydroxytabersonine
3.1.1 Yeast Culture
3.1.2 Product Isolation
3.2 Chemical Synthesis of (-)-16-Allyloxytabersonine
3.3 Chemical Synthesis of (-)-Na-Troc-Nb-Oxypachysiphine
3.4 Production of (-)-Melodinine K
4 Notes
References
Chapter 9: RNA-seq Analysis of Monoterpene Indole Alkaloid Biosynthetic Pathway Elucidation in Catharanthus roseus
1 Introduction
2 Materials
2.1 Software and Packages (See Note 1)
3 Methods
3.1 Reference Transcriptome Assembly
3.1.1 Obtaining RNA-seq Reads and Read Preprocessing
3.1.2 Transcriptome Assembly
3.1.3 Transcriptome Annotation
3.2 Exploratory Transcriptome Analysis
3.2.1 Differential Gene Expression Analysis
3.2.2 Pathway-Level Coexpression Network
3.3 Conclusion
4 Notes
References
Chapter 10: Predicting Monoterpene Indole Alkaloid-Related Genes from Expression Data with Artificial Neural Networks
1 Introduction
2 Material
2.1 Genomic Resources
2.2 Softwares and Packages
3 Methods
3.1 Load Data
3.2 Define Labels
3.3 Define Training Dataset
3.4 Train Network
3.5 Evaluation
3.6 Predict Labels and Evaluate
3.7 Optimization
3.8 Conclusion
4 Notes
References
Chapter 11: Discovery and Characterization of Oxidative Enzymes Involved in Monoterpenoid Indole Alkaloid Biosynthesis
1 Introduction
2 Materials
2.1 Software/Packages
2.2 Plant RNA Extraction and cDNA Synthesis
2.3 Candidate Genes Coning into the Expression Vector
2.4 Yeast Culture and Transformation
2.5 Enzymatic Assays
2.6 UPLC-MS/MS Analysis
2.7 Enzymatic Product Purification for NMR Analysis
3 Methods (See Note 1)
3.1 Candidate Gene Identification from Genomic and Transcriptomic Datasets (See Note 2)
3.1.1 Expression Analysis
Correlation Coefficients from Differential Expression Analysis
Correlation Coefficients from Coexpression Analysis (See Note 3)
Hierarchical Clustering (See Notes 3 and 4)
Self-Organizing Maps
3.1.2 Homology Search
3.1.3 Identification of Physically-Clustered Biosynthetic Genes
3.2 Biochemical Characterization of Candidate Genes in Yeast
3.2.1 Plant RNA Extraction and cDNA Synthesis (See Note 5)
3.2.2 Cloning for Yeast Expression (See Notes 6 and 7)
3.2.3 Yeast Transformation (See Notes 8 and 9)
3.2.4 In Vivo Activity Assays (See Notes 10-14)
3.2.5 In Vitro Activity Assays (See Notes 15 and 16)
3.2.6 Product Identification
UPLC-MS/MS Analysis
Product Purification and NMR Analysis
4 Notes
References
Chapter 12: Ancestral Sequence Reconstruction for Exploring Alkaloid Evolution
1 Introduction
2 Materials
2.1 Public Databases and Resources
2.2 Software
3 Methods
3.1 Sequence Selection
3.1.1 Amino Acids or Nucleotides?
3.1.2 Quality Control
3.2 Obtaining Sequences
3.3 Multiple Sequence Alignment
3.3.1 Software
3.3.2 Curation
3.3.3 Phylogeny-Aware Alignment
3.4 Tree Inference
3.4.1 Types of Trees
3.4.2 Maximum Likelihood Trees
3.4.3 Fast Trees
3.4.4 Tree QC
3.5 Ancestral Sequence Reconstruction
3.5.1 Background
3.5.2 Substitution Models
3.5.3 Software
3.5.4 Indels
3.5.5 Dealing with Ambiguity
3.6 What Next?
3.6.1 Experimental Work
3.6.2 Selection Analysis
3.7 Conclusion
References
Chapter 13: Generating an EMS Mutant Population and Rapid Mutant Screening by Thin-Layer Chromatography Enables the Studies of...
1 Introduction
2 Materials
2.1 Plant Cultures
2.2 Chemicals and Supplies
2.3 Other Devices
3 Methods
3.1 Generating the EMS Population M2
3.2 Preparing C. roseus Leaf Extracts (See Note 4)
3.3 Screening Mutant Population by TLC
4 Notes
References
Chapter 14: TARGETing Transcriptional Regulation in the Medicinal Plant Catharanthus roseus
1 Introduction
2 Materials
2.1 Plasmid Preparation
2.2 Isolation of Protoplasts
2.3 Transformation of Protoplasts Mediated by PEG and Activation of the GR Receptor Tag
2.4 FACS of Protoplasts and RNA Extraction
3 Method
3.1 Plasmid Preparation
3.2 Isolation of Protoplasts
3.3 Transformation of Protoplasts Mediated by PEG and Activation of the GR Receptor Tag (See Note 9)
3.4 FACS of Transformed Leaf Protoplasts and RNA Extraction
4 Notes
References
Chapter 15: Identification and Characterization of Transcription Factors Regulating Terpenoid Indole Alkaloid Biosynthesis in ...
1 Introduction
2 Materials
2.1 Co-expression Analysis
2.2 Databases for in Silico Analysis
2.3 C. roseus Hairy Roots
2.4 C. roseus Flower Infiltration
2.5 Electrophoretic Mobility Shift Assay (EMSA)
3 Methods
3.1 Identification of TFs
3.1.1 Co-expression Analysis
3.1.2 Identification of cis-Regulatory Motifs in Gene Promoters
The PLACE Database
The JASPAR Database
3.2 Characterization of TFs
3.2.1 Agrobacterium-Mediated Transformation of C. roseus Hairy Roots
3.2.2 Agrobacterium Infiltration of C. roseus Flowers
3.2.3 EMSA
4 Notes
References
Chapter 16: From Methylome to Integrative Analysis of Tissue Specificity
1 Introduction
2 Materials
2.1 Reagents and Consumables
2.2 Equipment
2.3 Genome Resource
2.4 Galaxy or R Packages
3 Methods
3.1 Ovation Methyl-seq Workflow
3.1.1 DNA Fragmentation
3.1.2 DNA Purification
3.1.3 End Repair
3.1.4 Ligation
3.1.5 Final Repair
3.1.6 Bisulfite Conversion (see Note 6)
3.1.7 Cleanup of Bisulfite Converted DNA
3.1.8 Determination of Optimal Amplification Cycles by qPCR
3.1.9 PCR Amplification
3.1.10 Quantitative and Qualitative Assessment of the Library
3.2 Sequencing
3.3 Processing of Sequencing Data (Fig. 3)
3.3.1 Quality Control of Sequencing Reads and Cleaning
3.3.2 Alignment and Methylation Calling
3.3.3 Identification of Differential Methylation: DMC and DMR (Fig. 3)
Workflow 1 (Galaxy, see Note 17)
Workflow 2 (DSS in R, see Note 18)
3.4 Data Exploration (see Note 18)
4 Notes
References
Chapter 17: Tagging and Capture of Prenylated CaaX-Proteins from Plant Cell Cultures
1 Introduction
2 Materials
2.1 Cell Treatment
2.2 Purification of Labeled CaaX-Proteins
3 Methods
3.1 Cell Treatment (see Note 1)
3.2 Purification of Labeled CaaX-Proteins
4 Notes
References
Chapter 18: EASI Transformation Protocol: An Agrobacterium-Mediated Transient Transformation Protocol for Catharanthus roseus ...
1 Introduction
2 Materials
2.1 Equipment and Supplies
2.2 Media Preparation
2.3 Biological Materials
2.4 Plasmids
3 Methods
3.1 C. roseus Seedling Preparation
3.2 Agrobacterium Preparation
3.3 Vacuum-Infiltration
4 Notes
References
Chapter 19: A Rapid and Efficient Vacuum-Based Agroinfiltration Protocol for Transient Gene Overexpression in Leaves of Cathar...
1 Introduction
2 Materials
2.1 Vectors Construction
2.2 Handling of Agrobacterium Cells
2.3 Preparation of Catharanthus roseus Plants
2.4 Vacuum Equipment
2.5 Macro/Microscopic Analysis
2.6 Extraction of Proteins and Western Blot
3 Methods
3.1 Construction of Vectors for Transient Expression
3.2 Agroinfiltration of C. roseus Plants
3.2.1 Preparation of C. roseus Plants
3.2.2 Preparation of Agrobacterium Strains for Agroinfiltration
3.2.3 Vacuum Infiltration
3.3 Validation of Transient Expression
3.3.1 Macroscopic Observation of GFP Fluorescence
3.3.2 Microscopic Observation of GFP Fluorescence
3.3.3 Protein Extraction
3.3.4 SDS-PAGE
3.3.5 Western Blot and Chemiluminescence Detection
4 Notes
References
Chapter 20: Transient Gene Expression in Catharanthus roseus Flower Petals Using Agroinfiltration
1 Introduction
2 Materials
2.1 Cloning Procedure
2.2 Agrobacterium Transformation and Growth
2.3 Agroinfiltration
2.4 Sampling
2.5 RNA Extraction
2.6 Metabolite Extraction
3 Methods
3.1 Cloning of Genes of Interest
3.2 Agrobacterium Transformation
3.3 Agroinfiltration
3.4 Sampling
3.5 RNA Extraction for Gene Expression Analysis
3.6 Metabolite Extraction for LC/MS
4 Notes
References
Chapter 21: De Novo Shoot Bud Induction from the Catharanthus roseus Leaf Explants and Agrobacterium tumefaciens-Mediated Tech...
1 Introduction
2 Materials
2.1 Biological Materials
2.2 Media Compositions
2.3 Culture Conditions
2.4 Equipment
3 Methods
3.1 Plant Material Preparation (See Note 1)
3.2 Agrobacterium-Mediated Genetic Transformation (Optional)
3.2.1 Agrobacterium Culture
3.2.2 Manual Pricking Inoculation (See Note 4)
3.2.3 Sonication-Assisted Agrobacterium Transformation (SAAT) Treatment (See Note 5)
3.3 Direct Shoot Bud Regeneration
4 Notes
References
Chapter 22: Agrobacterium-Mediated in Planta Transformation in Periwinkle
1 Introduction
2 Materials
2.1 Vector Construction
2.2 Agrobacterium Transformation
2.3 Agroinfiltration
2.4 Screening of Agro-Infected Plants
2.5 Alkaloid Analysis
3 Methods
3.1 Plant Expression Vector Construction
3.2 Transformation of Recombinant pBI121 into A. tumefaciens
3.3 Agro-Infection of C. roseus Seedlings
3.4 Evaluation of Transgenic Plants
3.5 Alkaloid Analysis
3.5.1 Secologanin
3.5.2 MIAs
4 Notes
References
Index