Plant secondary metabolism engineering : methods and protocols

Preface
Contents
Contributors
Chapter 1: Extraction and Analysis of Phenolic Compounds from Grape Berries
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Solutions
		2.3 Standards
		2.4 Equipment
	3 Methods
		3.1 Spectrophotometric Methods
			3.1.1 Folin-Ciocâlteu (F-C Reagent) for Determination of Total Phenolic Compounds
			3.1.2 Total Flavonoids and Anthocyanin Indexes of Grape Skin
			3.1.3 Determination of Monomer Flavanols by Vanillin Reaction
		3.2 Extraction and HPLC Analysis of Phenolic Compounds
			3.2.1 Extraction of Anthocyanins and Other Monomeric Phenols (Flavan-3-ols, Flavonols, and Hydroxycinnamates Derivatives)
			3.2.2 PA Extraction and Analysis
		3.3 High-Resolution Mass Spectrometry of Grape Polyphenols
	4 Notes
	References
Chapter 2: Construction of the UDP-Glucose Biosynthetic Enzyme Gene Coexpression Plasmid for Prunasin Production in Escherichi...
	1 Introduction
	2 Materials
		2.1 Growth Media
	3 Methods
		3.1 Preparation of Genomic DNA from E. coli JM109
		3.2 Molecular Cloning of glk, pgm, galU, and ppk
		3.3 Construction of UDP-Glucose Biosynthetic Enzyme Gene Coexpression Plasmids
		3.4 Transformation of E. coli
		3.5 Microbial Production of Prunasin
		3.6 Quantification of Prunasin Production
	4 Notes
	References
Chapter 3: Histochemical Localization of Phenolic Compounds and Reactive Oxygen Species in Eucalypt Microcuttings
	1 Introduction
	2 Materials
		2.1 Sectioning and Sample Preparation
		2.2 Total Phenolics Staining
		2.3 Lignin Staining
		2.4 Flavonoid Staining
		2.5 Superoxide Staining
		2.6 Hydrogen Peroxide Staining
		2.7 Imaging
		2.8 Stained Area Quantification
	3 Methods
		3.1 Sectioning and Sample Preparation
		3.2 Total Phenolics Staining
		3.3 Lignin Staining
		3.4 Flavonoid Staining
		3.5 Superoxide Staining
		3.6 Hydrogen Peroxide Staining
		3.7 Imaging
		3.8 Stained Area Quantification
	4 Notes
	References
Chapter 4: A Bioinformatics Tool for Efficient Retrieval of High-Confidence Terpene Synthases (TPS) and Application to the Ide...
	1 Introduction
		1.1 Overall Context
		1.2 Model Construction
	2 Materials
		2.1 Hardware, System, and Sequence Data
		2.2 Input, Software and Availability
	3 Method
		3.1 Tool Validation
		3.2 Application of search_TPS
	4 Notes
	References
Chapter 5: Use of Catharanthus roseus Cell Cultures for the Synthesis of Metal Nanoparticles
	1 Introduction
	2 Materials
		2.1 Initiation of Callus and Cell Suspension Cultures
		2.2 Synthesis of Silver Nanoparticles with Seed-Derived Callus Extract
		2.3 Gold Nanoparticle Synthesis with Cell-Free Suspension Culture Filtrate
	3 Methods
		3.1 Initiation of Callus Culture from Seeds
		3.2 Initiation of Cell Suspension from Seed-Derived Calluses
		3.3 Synthesis of Silver Nanoparticles with Seed-Derived Callus Extract
		3.4 Gold Nanoparticle Synthesis with Cell-Free Suspension Culture Filtrate
	4 Notes
	References
Chapter 6: Fungal Pigment-Assisted Silver Nanoparticle Synthesis and Their Antimicrobial and Cytotoxic Potential
	1 Introduction
	2 Materials
		2.1 Extracellular Pigment Production Media
		2.2 Microbial Cell Death Kinetics Study (Used to Compare Nanoparticles Effects with Those of a Standard Antibiotic Against a M...
		2.3 Anticancer Activity of Biogenic AgNPs
	3 Methods
		3.1 Media and Spore Preparation
		3.2 Extraction of Extracellular Pigment
		3.3 AgNPs Synthesis Using the Pigment
		3.4 Characterization of AgNPs by  TEM
		3.5 Particle Size Estimation from TEM Images Using ImageJ Fiji (Version 2.0.0-rc-68/1.52e)
		3.6 Microbial Cell Death Kinetics Study
		3.7 Cell Line Cultivation
		3.8 Anticancer Assay
		3.9 Assay for Calculation of the Half-Maximal Inhibitory Concentration (IC50) Value
		3.10 Data Fitting with a Four-Parameter Logistic Model Using ImageJ Fiji (Version 2.0.0-rc-68/1.52e)
	4 Notes
	References
Chapter 7: Nuclei Enrichment from Sugarcane Stems for Proteomics Analyses
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Nuclei Enrichment
		2.3 Western Blot
			2.3.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			2.3.2 Immunoblotting
	3 Methods
		3.1 Tissue Harvest and Storage
		3.2 Pulverization of the Stem Tissue
		3.3 Nuclei Enrichment from Stems
		3.4 Nuclear Protein Extraction
		3.5 Enrichment Assessment of Nuclei: Western Blotting
			3.5.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			3.5.2 Immunoblotting
	4 Notes
	References
Chapter 8: Visualization of Suberization and Lignification in Sugarcane
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Suberin Lytic Enzymatic Assay and Sudan IV Histochemical Assay
		2.3 Mechanically Induced Lignification Test and Lignin Determination
		2.4 Sugarcane Total RNA Extraction and Gene Expression
	3 Methods
		3.1 Suberin Lytic Enzymatic Assay
		3.2 Mechanically Induced Suberization Test
		3.3 Sudan IV Histochemical Assay
		3.4 Mechanically Induced Lignification Test
		3.5 Lignin Determination: Acetyl Bromide Method
		3.6 Sugarcane Total RNA Extraction
		3.7 Gene Expression Dynamics of Sugarcane Genes (See Note 10)
	4 Notes
	References
Chapter 9: Isolation of Promoters and Transcription Factors Involved in the Regulation of Lignin Biosynthesis in Saccharum Spe...
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
		2.3 Biological Material
	3 Methods
		3.1 Extraction of Genomic DNA
		3.2 Extraction of Total RNA and cDNA Synthesis
		3.3 Isolation of pShCAD8, pShCOMT and pShF5H Promoters
			3.3.1 Promoter pShCAD8
			3.3.2 Promoters: pShF5H and pShCOMT
		3.4 Isolation of Transcription Factors: ShMYB85 and ShMYB58/63
		3.5 Promoters (pShCAD8, pShF5H, and pShCOMT) and Transcription Factors (ShMYB85 and ShMYB58/63) Cloning
		3.6 Transactivation Assays in BY2 Protoplasts
	4 Notes
	References
Chapter 10: Soapbark Triterpenes: Quillaja brasiliensis Cell Culture Sapogenin and Free Sterol Analysis by GCMS
	1 Introduction
	2 Materials
		2.1 Plant Material
		2.2 Equipment
		2.3 Extraction of Sterols
		2.4 Extraction of Saponins
		2.5 Acid Hydrolysis for Free Sterol Analysis
		2.6 Acid Hydrolysis for Sapogenin Analysis
		2.7 Derivatization for GCMS Analysis
		2.8 GCMS Analysis
	3 Methods
		3.1 Extraction of Total Sterols
		3.2 Acid Hydrolysis for Free Sterols Analysis
		3.3 Extraction of Saponins
		3.4 Acid Hydrolysis for Sapogenin Analysis
		3.5 Derivatization for GCMS Analysis
		3.6 GCMS Analysis
		3.7 Quantification and Data Analysis
	4 Notes
	References
Chapter 11: Generation of Stable Catharanthus roseus Hairy Root Lines with Agrobacterium rhizogenes
	1 Introduction
	2 Materials
		2.1 Sterilization of Seeds and Growth of Young Plants
		2.2 Electroporation of A. rhizogenes and Preparation of Glycerol Stocks
		2.3 Infection of 1- to 2-Month-Old Plants with A. rhizogenes
		2.4 Excising and Plating Roots for Agrobacterium Elimination and Selection of Transgenic Hairy Roots
		2.5 Transfer of Roots to Liquid Media and Subculture Protocol
		2.6 Validation of Genomic Integration of T-DNA with  PCR
	3 Methods
		3.1 Sterilization of Seeds and Cultivation in Magenta Boxes
			3.1.1 Seed Sterilization
			3.1.2 Planting and Maintenance of Seedlings
		3.2 Preparation of A. rhizogenes
			3.2.1 Transformation of A. rhizogenes with Engineered Binary Vector
			3.2.2 A. rhizogenes Cultivation in Preparation for Infecting C. roseus Plants
			3.2.3 Infection of Young Plants with A. rhizogenes
		3.3 Excision and Selection of Transgenic Root Tissue from Whole Plants
		3.4 Cultivation and Stabilization of Roots in Liquid Media
		3.5 Verification of T-DNA Genomic Integration with  PCR
	4 Notes
	References
Chapter 12: Analyzing the Distribution of Specialized Metabolites from Plant Native Tissues with Laser Desorption Low-Temperat...
	1 Introduction
	2 Materials
		2.1 Biological Samples
		2.2 Additional Materials
		2.3 Laser Desorption Low-Temperature Plasma Mass Spectrometry Imaging System
		2.4 Image Analysis
	3 Methods
		3.1 Setting up the Imaging System
		3.2 Sample Preparation
		3.3 Data Acquisition
		3.4 Image Data Processing and Analysis
	4 Notes
	References
Chapter 13: Virus-Induced Gene Silencing as a Tool to Study Regulation of Alkaloid Biosynthesis in Medicinal Plants
	1 Introduction
	2 Material
		2.1 Plant Material
		2.2 Plasmid and Primers
		2.3 Enzymes and Chemicals
		2.4 Infiltration Buffer
		2.5 Media Preparations
	3 Methods
		3.1 Seed Germination
		3.2 Construction of VIGS Vectors
		3.3 Preparation of Chemically (CaCl2) Competent Agrobacterium Cells
		3.4 Agrobacterium Transformation
		3.5 Inoculation of C. roseus Plants with Agrobacterium
		3.6 Leaf Sample Collection for Gene Expression and Alkaloid Analysis
		3.7 RNA Isolation and Gene Expression Analysis
		3.8 Alkaloid Extraction and Analysis
	4 Notes
	References
Chapter 14: Cyclotide Isolation from Psychotria brachyceras and Psychotria leiocarpa
	1 Introduction
	2 Materials
		2.1 Confirmation of Cyclotide Presence in Plant Material
		2.2 Cyclotide Extraction
		2.3 Cyclotide Isolation
			2.3.1 Solid-Phase Extraction (SPE)
			2.3.2 High Performance Liquid Chromatography (HPLC)
	3 Methods
		3.1 Confirmation of Cyclotide Presence in Plant Material
			3.1.1 Sample Preparation for Searching Native Peptides
			3.1.2 Sample Preparation for Searching Reduced and Alkylated Peptides
			3.1.3 Sample Preparation for Searching Enzymatic Digested Peptides
			3.1.4 MALDI Analysis
		3.2 Cyclotide Extraction
		3.3 Cyclotide Isolation
			3.3.1 Solid-Phase Extraction (SPE)
			3.3.2 High-Performance Liquid Chromatography (HPLC)
	4 Notes
	References
Chapter 15: Formulation of IMXQB: Nanoparticles Based on Quillaja brasiliensis Saponins to be Used as Vaccine Adjuvants
	1 Introduction
	2 Materials
		2.1 Formulation of IMXQB Nanoparticles
		2.2 High-Resolution Transmission Electronic Microscopy and Dynamic Light Scattering (DLS)
	3 Methods
		3.1 Formulation of IMXQB Nanoparticles by Dialysis Method
		3.2 Verification Steps
			3.2.1 Visualization of the Nanoparticles by High-Resolution Transmission Electron Microscopy (HR-TEM)
			3.2.2 Particle Size Measurement
			3.2.3 Electrophoretic and Zeta Potential Measurements
	4 Notes
	References
Chapter 16: Isolation of Specialized Plant Cells by Fluorescence-Activated Cell Sorting
	1 Introduction
	2 Materials
		2.1 Plant Materials
		2.2 Isolation of Protoplasts
		2.3 FACS of Leaf Protoplasts
	3 Methods
		3.1 Isolation of Protoplasts
		3.2 FACS of Leaf Protoplasts
	4 Notes
	References
Chapter 17: X-Ray Computed Tomography for 3D Anatomical Analysis of Resin Ducts
	1 Introduction
	2 Materials
		2.1 Stem Sampling, Prescanning Preparation, and Scanning
		2.2 Materials for Digital Image Processing and Visualization
	3 Methods
		3.1 Stem Sampling and Handling in the Field
		3.2 Prescanning Sample Preparation
		3.3 Specimen Mounting and Scanning
		3.4 Digital Image Processing, Reconstruction and Visualization
		3.5 Software Setup, Segmentation of Resin Ducts, Quantitative Analysis and Data Visualization
			3.5.1 Software Setup
				Plugin Installation
				Image loading
				Preprocessing
				Binarization
			3.5.2 Segmentation
			3.5.3 Quantification of Resin Ducts
			3.5.4 Data Visualization in 3D and Use of 3D Suite-Dataviewer
			3.5.5 CTVox for Mobile App or  Ipad
	4 Notes
	References
Chapter 18: Untargeted Metabolomic Analysis of Nonvolatile and Volatile Glucosinolates in Brassicaceae
	1 Introduction
	2 Materials
		2.1 Glucosinolates (GLSs) Standard Solutions
		2.2 Isothiocyanates (ITCs) Standard Solutions
		2.3 Mobile Phase Solutions Used in LC-MS
		2.4 Equipment
			2.4.1 Equipment for Analysis of GLSs
			2.4.2 Equipment for Analysis of ITCs
	3 Methods
		3.1 Vegetable Samples
		3.2 Untargeted Analysis of Glucosinolates
			3.2.1 UHPLC-Q-TOF Setup
			3.2.2 Sample Extraction
			3.2.3 Data Acquisition and Analysis
		3.3 Untargeted Analysis of Volatile Metabolites (Including ITCs)
			3.3.1 GC-Q-TOF Setup
			3.3.2 Volatile Metabolites Extraction
			3.3.3 Data Acquisition and Analysis
		3.4 Stability Evaluation of the Analytical Platform
			3.4.1 QCs for LC-MS
			3.4.2 QCs for GC-MS
	4 Notes
	References
Chapter 19: Methods of Mimosine Extraction from Leucaena leucocephala (Lam.) de Wit Leaves
	1 Introduction
	2 Materials
		2.1 Extracts from Leaves of L. leucocephala
			2.1.1 Pulverized Leaf Maceration
			2.1.2 Whole-Leaf Maceration
		2.2 Analysis
		2.3 Reagents and Solutions
	3 Methods
		3.1 Pulverized Leaf Maceration in Boiling Water (HPLM)
		3.2 Pulverized Leaf Maceration at Room Temperature (CPLM)
		3.3 Whole-Leaf Maceration (WLM)
		3.4 HPLC Sample Preparation
	4 Notes
	References
Chapter 20: Microbial Production, Extraction, and Quantitative Analysis of Isoprenoids
	1 Introduction
	2 Materials
		2.1 Production and Quantification of Monoterpenes, Sesquiterpenes, and diterpenes in Yeast
			2.1.1 Reagents and Material
			2.1.2 Analytical Devices and Equipment
			2.1.3 Growth Media
		2.2 Production and Quantification of Triterpenoids in Yeast
			2.2.1 Reagents and Material
			2.2.2 Analytical Devices and Equipment
			2.2.3 Growth Media
		2.3 Quantification of Pigment Production in Cyanobacteria
			2.3.1 Reagents and Material
			2.3.2 Analytical Devices and Equipment
			2.3.3 Growth Media
	3 Methods
		3.1 Yeast Cultivation and Sampling for Monoterpene, Sesquiterpene and Diterpene Quantification
			3.1.1 Growth Media Preparation
			3.1.2 Set up Two-Phase Flask Cultivation
			3.1.3 Sample Preparation for Terpene Quantification
		3.2 Yeast Cultivation and Sampling for Triterpenoid Quantification
			3.2.1 Growth Medium Preparation
			3.2.2 Shake Flask Cultivations
			3.2.3 Sample Preparation for HPLC-CAD Analysis
		3.3 Cyanobacterial Cultivation and Sampling for Carotenoid Quantification
			3.3.1 Growth Media Preparation
			3.3.2 Shake-Flask Cultivations
			3.3.3 Sample Preparation for HPLC Analysis
		3.4 Isoprenoid Quantification Via HPLC Analysis
			3.4.1 Mono-, Sesqui-, and Diterpenoid Quantification by HPLC-DAD
			3.4.2 Triterpenoid Quantification by HPLC-CAD
			3.4.3 Carotenoid Quantification by HPLC-DAD
	4 Notes
	References
Index