Cell Components (Molecular Methods of Plant Analysis)

Modern Methods of Plant Analysis When the handbook Modern Methods of Plant Analysis was first introduced in 1954 the considerations were 1. the dependence of scientific progress in biology on the improvement of existing and the introduction of new methods; 2. the inavailability of many new analytical methods concealed in specialized journals not normally accessible to experimental plant biologists; 3. the fact that in the methods sections of papers the description of methods is frequently so compact, or even sometimes so incomplete, that experiments are difficult to reproduce. These considerations still stand today. The series was highly successful, seven volumes appearing between 1956 and 1964. Since today there is still a demand for the old series, the publisher has decided to resume publication of Modern Methods of Plant Analysis. It is hoped that the New Series will be as acceptable to those working in plant sciences and related fields as the early volumes undoubtedly were. It is difficult to single out the major reasons for success of any publication, but we believe that the methods published in the first series were up-to-date at the time and the descriptions as applied to plant material so complete in themselves that there was little need to consult other publications.

Cell-Wall-Isolation, General Growth Aspects.- 1 Introduction.- 2 Isolation Procedures.- 2.1 Cell Breakage.- 2.2 Cell-Wall Recovery.- 2.3 Removal of Contaminants.- 3 Composition and Ultrastructure of Plant Cell Walls.- 3.1 Chemical Composition of Plant Cell Walls.- 3.1.1 Standard Extraction Procedures.- 3.1.2 Analysis of Polysaccharide Fractions.- 3.1.2.1 Chemical Methods.- 3.1.2.2 Physical Methods.- 3.2 Supramolecular Organization of Plant Cell Walls.- 3.2.1 Morphological Observations.- 3.2.2 Selective Staining of Polysaccharides.- 3.2.2.1 Visualization of Esterified Carboxyl Groups.- 3.2.2.2 Detection of Acidic Functions.- 3.2.2.3 Periodic Oxidation of Glycol Groups.- 3.2.3 Visualization of Lignin.- 3.2.4 Identification of Wall Components by Means of Affinity Methods.- 3.2.5 Detection and Estimation of Cations.- 3.2.6 Ultracryotomy.- 4 Properties of Plant Cell Walls.- 4.1 Exchange Properties of Plant Cell Walls.- 4.2 Enzymatic Properties.- 4.2.1 Cytochemical Investigations.- 4.2.1.1 Cell-Wall Phosphatase Activities.- 4.2.1.2 Cell-Wall Peroxidase Activities.- 4.2.2 Biochemical Investigations.- 4.2.2.1 Properties of Immobilized and Solubilized Cell-Wall Enzymes.- 4.2.2.2 Biological Functions.- 4.3 Mechanical Properties.- 5 Growth Aspects.- 5.1 Cell-Wall Loosening.- 5.1.1 Wall-Loosening-Inducing Agents.- 5.1.2 Nature of the Broken Bonds.- 5.2 Deposition of Wall Material.- 5.3 Growth Direction.- References.- Cell-Wall Chemistry, Structure and Components.- 1 Introduction.- 2 Histochemical Analysis of Cell Walls.- 2.1 Specific Stainings.- 2.2 Staining with Fluorescent Brightener.- 2.3 Anisotropy Test.- 2.4 Selective Dissolution.- 2.4.1 Alkali Treatment.- 2.4.2 Cuprammonium Solution (Schweitzer’s Reagent) Treatment.- 2.4.3 Enzymatic Digestion.- 3 Quantitative Analysis of Cell Walls.- 3.1 Plant Materials.- 3.1.1 Pure Culture.- 3.1.2 Synchronous Culture.- 3.1.3 Harvesting of Cells.- 3.2 Measurement of Cell Growth.- 3.3 Preparation and Fractionation of Cell Walls.- 3.3.1 Disruption of Cells.- 3.3.2 Separation and Purification of Cell Walls.- 3.3.3 Fractionation of Cell Walls.- 3.4 Quantitative Analysis of Whole Cell Walls.- 3.4.1 Gravimetry.- 3.4.2 Turbidimetry.- 3.4.3 Colorimetry.- 4 Qualitative Analysis of Cell-Wall Materials.- 4.1 Acid Hydrolysis.- 4.2 Enzymatic Hydrolysis.- 5 Chromatographic Analysis of Cell-Wall Constituents.- 5.1 Thin-Layer Chromatography.- 5.1.1 Neutral Sugars and Uronic Acids.- 5.1.2 Amino Acids and Amino Sugars.- 5.1.3 Thin-Layer Chromatographic Analyses of the Constituents of Chlorella Cell Walls.- 5.2 Liquid Chromatography.- 5.2.1 Amino Acids and Amino Sugars.- 5.2.2 Neutral Sugars.- References.- Protoplasts―for Compartmentation Studies.- 1 Introduction.- 2 Advantages of the Use of Protoplasts for Compartmentation Studies.- 3 Protoplast Isolation and Its Effect on Cellular Metabolism.- 3.1 Isolation Procedures.- 3.2 Effect of Isolation pH.- 3.3 Effect of Plasmolysis.- 3.4 Effect of Enzyme Contaminants.- 4 Protoplast Lysis.- 5 Protoplast Fractionation.- 5.1 Density Gradient Fractionation.- 5.2 Rapid Fractionation Procedures.- 6 Methods to Relate Protoplast Activity to That of Intact Tissue.- 7 Concluding Remarks.- References.- The Marker Concept in Cell Fractionation.- 1 Introduction.- 2 The Marker Concept.- 2.1 Basic Concepts.- 2.2 Types of Marker.- 2.2.1 Morphological.- 2.2.2 Cytochemical.- 2.2.3 Biochemical.- 3 Preservation of Marker Enzyme Activity During Cell Disruption.- 3.1 Choice of Material.- 3.2 Homogenization Procedure.- 3.3 Use of Additives in the Homogenization Medium.- 3.4 Gel Filtration to Remove Soluble Hydrolytic Activity.- 4 Methods Used to Separate Markers.- 4.1 General Approaches to Cell Fractionation.- 4.2 Differential Centrifugation.- 4.2.1 Preparative vs. Analytical Cell Fractionation.- 4.2.2 Need for Quantitation.- 4.2.3 Problems with Complete Quantitation and Interpretation of Data.- 4.3 Linear Density Gradient Centrifugation.- 4.3.1 Density Gradient Material.- 4.3.2 Pelleted vs. Unpelleted Overlays.- 4.3.3 Soluble Enzyme Contamination in Gradients.- 4.3.4 Equilibrium Density Centrifugation (Isopycnic Conditions).- 4.3.5 Other Factors Which Influence Marker Enzyme Profiles Across a Gradient.- 4.3.6 Need for Quantitation and Lack of Negative Marker Activity.- 5 Concluding Remarks.- References.- Plasma Membranes.- 1 Introduction.- 2 Theory of Phase Partition.- 2.1 The Phase System.- 2.2 Partitition of Membrane Particles.- 2.3 Effects of Polymer Concentrations.- 2.4 Effects of Salts.- 2.5 Multistep Procedures.- 3 Experimentals.- 3.1 Chemicals.- 3.2 Preparation Procedure.- 4 Purity of the Preparations.- 4.1 Specific Staining.- 4.2 K+-Stimulated, Mg2+-Dependent ATPase.- 4.3 Glucan Synthetase II.- 4.4 Light-Reducible b-Cytochrome.- 4.5 Markers for Contaminants.- 5 Protein and Lipid Composition.- 6 Surface Properties of the Isolated Vesicles.- References.- Vacuoles.- 1 Introduction.- 2 Methods of Isolation.- 2.1 Isolation of Vacuoles from Meristematic Tissues.- 2.2 Isolation of Vacuoles from Mature Plant Tissue.- 2.2.1 Isolation of Mature Vacuoles from Protoplasts―Methods pre 1981.- 2.2.2 Isolation of Mature Vacuoles from Protoplasts―Methods post 1981.- 2.2.3 Isolation of Mature Vacuoles Directly from Tissue―Methods pre 1981.- 2.2.4 Isolation of Mature Vacuoles Directly from Tissue―Methods post 1981.- 2.2.5 Preparation of Lutoids from Hevea Latex.- 2.2.6 Comments on Methods for Isolating Vacuoles from Higher Plants.- 2.2.7 Isolation of Proton-Pumping Vesicles.- 2.2.8 Preparation of Vacuoles from Yeast Neurospora.- 3 Isolation of Tonoplast and Tonoplast Markers.- 4 Comments on Physiological Functions.- 5 Concluding Remarks.- References.- Protein Bodies.- 1 Introduction.- 2 Special Consideration in Isolation of Protein Bodies.- 3 Nonaqueous Preparation in Glycerol.- 4 Nonaqueous Preparation in Hexane and Carbon Tetrachloride.- 5 Aqueous Preparation in Sources Gradients.- 6 Subfractionation of Isolated Protein Bodies.- 7 Analyses.- References.- Lipid Bodies.- 1 Introduction.- 2 Ontogeny.- 3 Isolation.- 4 Markers of Lipid Bodies.- 5 Assays.- 5.1 Fluorometric Assay.- 5.2 Colorimetrie Assay.- References.- Chloroplasts as a Whole.- 1 Introduction.- 2 Considerations of Integrity and Purity.- 3 Chloroplasts from Protoplasts.- 4 The Use of Silica Sols in Density Gradient Purification of Chloroplasts.- 5 General Notes on Isolation Procedures.- 6 Specific Isolation Protocols.- 6.1 Higher Plants.- 6.1.1 C3 Plants.- 6.1.2 C4 Plants.- 6.1.3 CAM Plants.- 6.2 Algae.- 6.2.1 Volvocales.- 6.2.2 (Ceramiaceae, Rhodophyta)―Griffithsiamonilis.- 6.2.3 Siphonales.- 6.2.4 (Xanthophyceae) Bumilleriopsisfiliformis.- 6.2.5 (Euglenophyceae) Euglenagracilis.- 7 Additional Comments on Chloroplast Isolation.- 8 Abbreviations.- References.- Purification of Inner and Outer Chloroplast Envelope Membranes.- 1 Introduction.- 2 General Considerations.- 3 The Procedure.- 3.1 Reagents and Equipment.- 3.1.1 Solutions.- 3.1.2 Materials.- 3.2 Growth of Peas and Purification of Intact Chloroplasts.- 3.3 Purification of Inner and Outer Envelope Membranes.- 4 Properties of the Isolated Membranes.- 4.1 Purity.- 4.1.1 Cross-Contamination by Envelope Membranes.- 4.1.2 Contamination by Thylakoids.- 4.1.3 Contamination by Stroma.- 4.2 Other Properties.- 5 Modifications of the Procedure.- 5.1 Alternate Methods of Chloroplast Rupture.- 5.2 Purification Subsequent to Rupture.- 5.3 Application to Other Tissues.- 6 Other Procedures.- References.- The Major Protein of Chloroplast Stroma, Ribulosebisphosphate Carboxylase.- 1 Introduction.- 2 Characteristics of RuBP Carboxylase.- 2.1 Molecular Arrangement and Physical Structure of Subunits.- 2.2 Molecular Structure.- 2.3 Biosynthesis and Assembly of Subunits.- 2.3.1 Large Subunit.- 2.3.2 Small Subunit.- 2.3.3 Subunit Heterogeneity.- 2.3.4 Coordinate Control of Subunit Synthesis.- 2.4 Catalytic Mechanism.- 2.4.1 Activation and Role of Mg2 +.- 2.4.2 Carboxylation of RuBP.- 2.4.3 Oxygenation of RuBP.- 2.4.4 Localization of Catalytic and Activator Site.- 3 Practical Aspects.- 3.1 Purification.- 3.1.1 Summary of Techniques.- 3.1.2 Interfering Compounds.- 3.1.3 Choice of Extraction Buffer and Grinding Procedures.- 3.1.4 Protein Determination.- 3.1.5 Example: RuBP Carboxylase from Soybean Leaves.- 3.2 Assay.- 3.2.1 Substrates.- 3.2.2 Activation of RuBP Carboxylase.- 3.2.3 Continuous Spectrophotometry Assay for RuBP Carboxylase Activity.- 3.2.4 Discontinuous Assays for RuBP Carboxylase Activity.- 3.2.4.1 Radiochemical Assay with [14C]NaHCO3.- 3.2.4.2 Radiochemical Assay Using [14C]NaHCO3 and [l-3H]RuBP.- 3.2.4.3 Discontinuous Assay Using Nonlabeled Substrates.- 3.2.5 Assays for RuBP Oxygenase.- 3.2.6 Kinetic Parameters of RuBP Carboxylase and RuBP Oxygenase.- 4 Conclusion.- References.- The Chloroplast Thylakoid Membrane―Isolation, Subfractionation and Purification of Its Supramolecular Complexes.- 1 Introduction.- 2 Function and Organization of the Thylakoid Membrane.- 3 Isolation of Thylakoid Membranes.- 4 Thylakoid Membrane Subfractionation.- 4.1 Photosystem I Stroma Lamellae Thylakoids.- 4.2 Photosystem II Oxygen Evolving Thylakoid Preparations.- 4.2.1 Isolation by Press Treatment and Phase Partition.- 4.2.2 Isolation by Detergent Fractionation.- 4.2.3 Choice of Preparation.- 4.3 Separation of Inside-Out and Right-Side-Out Thylakoid Vesicles with the Same Composition.- 5 Isolation of Thylakoid Supramolecular Complexes.- 5.1 The Photosystem I Complex and the Light-Harvesting Complex of Photosystem II (LHC II).- 5.2 The Light-Harvesting Complex of Photosystem I (LHC I).- 5.3 The Inner Core Complex of Photosystem II (CC II).- 5.4 The Cytochrome b/f Complex.- 5.5 The ATP Synthase (CF0-CF1).- References.- The Isolation and Characterization of Nongreen Plastids.- 1 Introduction.- 2 The Terminology of Nongreen Plastids.- 2.1 Proplastids.- 2.2 Etioplasts.- 2.3 Chromoplasts.- 2.4 Amyloplasts.- 2.5 Leucoplasts.- 2.6 Other Nongreen Plastids.- 3 Basics of Plastid Isolation and Separation.- 3.1 Experimental Design.- 3.2 Isolation Medium.- 3.3 Tissue Disruption.- 3.4 General Methods of Chloroplast Isolation.- 4 Isolation of Nongreen Plastids from Developing Ricinus Endosperm.- 4.1 Rate-Zonal Sedimentation.- 4.1.1 Protocol.- 4.1.2 Analysis.- 4.1.3 Comments.- 4.2 Isopycnic Banding on Linear Sucrose Gradients.- 4.2.1 Protocol.- 4.2.2 Comments.- 4.3 Rate-Zonal Sedimentation on Linear Sucrose-Magnesium Co-Gradients.- 4.3.1 Protocol.- 4.3.2 Comments.- 4.4 Rate-Zonal Sedimentation on Discontinuous Sucrose Gradients.- 4.4.1 Protocol.- 4.4.2 Comments.- 4.5 Rate-Zonal Sedimentation on Discontinuous Percoll Gradients.- 4.5.1 Protocol.- 4.5.2 Comments.- 4.6 Nonaqueous Methods.- 4.6.1 Isopycnic Banding on Linear Hexane-CCl Gradients.- 4.6.2 Silicon Oil Centrifuged Filtration.- 4.7 Noncentrifugal Methods.- 4.7.1 Gel Permeation.- 4.7.1.1 Materials.- 4.7.1.2 Protocol.- 4.7.1.3 Comments.- 4.7.2 Phase Partition.- 4.7.3 Unit-Gravity Sedimentation.- 5 Metabolic Capabilities of Ricinus Endosperm Plastids.- 5.1 Glycolysis, the Pentose-Phosphate Pathway and Fatty Acid Synthesis.- 5.2 The Calvin Cycle.- 5.3 Nitrogen Metabolism.- 5.4 Terpenoid Metabolism.- 6 Composition and Biochemical Properties.- 6.1 Structure.- 6.2 Protein Composition.- 6.3 Membranes.- 6.4 Nucleic Acids.- 7 Future Prospects.- References.- Mitochondria.- 1 Introduction.- 2 Preparation for DNA Analysis.- 2.1 Cytoplasmic Male Sterility and Structure ot Mitochondrial DNA.- 2.2 Isolation of Mitochondria for DNA Preparation.- 2.3 Preparation of Mitochondrial DNA.- 2.4 Electrophoresis of Mitochondrial DNA.- 2.5 Restriction Analysis of Mitochondrial DNA.- 2.6 Notes on Mitochondrial DNA Studies.- 3 Preparation of Intact Mitochondria for Oxidative Studies.- 3.1 Introduction.- 3.2 Mitochondrial Preparation and Purification.- 3.3 Tests for Integrity of Mitochondria.- 3.3.1 Succinate: Cytochrome c Reductase.- 3.4 Tests for Integrity.- 3.4.1 Measurement for Oxygen Consumption for Respiratory Control and P/O Ratios.- 3.5 Notes on the Methods.- References.- Endoplasmic Reticulum.- 1 Introduction.- 2 Structure and Organization of the ER.- 3 Interactions Between Tubular and Cisternal ER.- 3.1 Role in Protein Transport.- 3.2 Role in Cell Division.- 4 Synthesis and Degradation of ER.- 4.1 Membrane Proteins.- 4.2 Membrane Lipids.- 5 Isolation and Characterization of ER.- 5.1 Isolation Media.- 5.2 Tissue Homogenization.- 5.3 Organelle Isolation.- 5.3.1 Molecular Sieve Chromatography.- 5.3.2 Differential Centrifugation.- 5.3.3 Density Gradient Centrifugation.- 5.4 Identification of ER Membranes.- 5.4.1 Magnesium Shift.- 5.4.2 Marker Enzymes.- 5.4.3 Auxin Binding.- 5.4.4 Calcium Transport.- 5.4.5 Structural Proteins.- 5.5 Concluding Remarks.- References.- Polyribosomes.- 1 Introduction.- 2 Isolation of Polysomes from Plant Cells.- 2.1 Factors that Affect the Stability and Recovery of Polyribosomes.- 2.2 Tissue Preparation.- 2.3 Subcellular Fractionation and Polysome Isolation.- 3 Purification and Analysis of Polyribosomes.- 3.1 Sucrose Gradient Centrifugation.- 3.2 Purification of Polysomes with Discontinuous Sucrose Gradients.- 3.3 Analysis of Polyribosome Profiles.- 4 Polyribosome Extraction Buffers.- 4.1 pH.- 4.2 Potassium Chloride.- 4.3 Magnesium Chloride.- 4.4 Reducing Agents.- 4.5 Chelation of Divalent Metals.- 4.6 Proteinase K.- 4.7 Other Ribonuclease Inhibitors.- 5 Uses of Purified Polyribosomes.- 5.1 Changes in Protein Synthetic Activity.- 5.2 In Vitro Protein Synthesis.- 5.3 Purification of mRNA’s.- 5.4 Subcellular Distribution of mRNA’s.- References.- The Nucleus―Cytological Methods and Isolation for Biochemical Studies.- 1 Introduction.- 2 Structure of the Plant Nucleus and Implications for Nuclear Isolation and Staining.- 3 Cytology.- 3.1 Nuclei of Whole Cells.- 3.1.1 Feulgen Microspectrophotometric Methods.- 3.1.2 Microfluorometry DNA Determination.- 3.2 Staining Nuclei During Isolation.- 3.3 Nucleolus Staining.- 3.4 Chromosome Staining.- 3.5 Other Nuclear Stains.- 4 Isolation of Plant Nuclei―General.- 4.1 Isolation of Plant Nuclei―Methods.- 4.1.1 Nuclei from Tobacco Callus Cultures for RNA Synthesis Studies.- 4.1.2 Nuclei from Tobacco Cells in Culture―for General Purpose Studies.- 4.1.3 Nuclei from Soybean Cells―for DNA Studies.- 4.1.4 Plant-Root Nuclei―for DNA Analysis.- 5 Summary.- References.- Microtubules.- 1 Introduction.- 2 Extraction of Microtubule Proteins.- 3 Purification of Tubulin and MAP’s.- 3.1 DEAE-Sephadex Ion Exchange Chromatography.- 3.2 Phosphocellulose Chromatography.- 3.3 Affinity Chromatography.- 3.4 Cycles of Polymerisation and Depolymerisation.- 3.4.1 Pre-Conditions for Microtubule Assembly.- 3.4.2 Microtubule Assembly in the Presence or Absence of Glycerol.- 3.4.3 The Dynamics of Polymerisation and the Use of Taxol.- 3.4.4 Co-Polymerisation.- 4 Fractionation and Identification of Tubulin by SDS-PAGE.- 5 Colchicine-Binding Assay for Tubulin.- 6 Immunochemical Methods of Analysis.- 6.1 Radioimmunoassay.- 6.2 Enzyme-Linked Immunosorbent Assay (ELISA).- 6.3 Antibody Purification of Antigen-Affinity Column.- 6.4 Western Blots.- 7 Concluding Remarks.- References.