Plant Cell Wall Analysis

Fractionation of Cell Wall Components.- 1 Introduction.- 2 Polysaccharides.- 2.1 Precipitation Reactions.- 2.1.1 Adjustment of pH.- 2.1.2 Precipitation with Organic Solvents.- 2.1.3 Precipitation with Inorganic Salts.- 2.1.4 Precipitation with Iodine.- 2.1.5 Precipitation with Ionic Detergents.- 2.2 Chromatography.- 2.2.1 Size Chromatography.- 2.2.2 Ion Exchange Chromatography.- 2.2.3 Affinity Chromatography.- 2.3 Electrophoresis.- 2.3.1 Moving Boundary Electrophoresis.- 2.3.2 Paper Electrophoresis.- 2.4 Miscellaneous Polysaccharide Methods.- 2.4.1 Cellulose.- 2.4.2 Polysaccharide Derivatives.- 3 Proteins and Glycoproteins.- 3.1 Precipitation Reactions.- 3.1.1 Precipitation with Ammonium Sulphate.- 3.1.2 Precipitation with Trichloroacetic Acid.- 3.2 Chromatography.- 3.2.1 Size Chromatography.- 3.2.2 Ion Exchange Chromatography.- 3.3 Electrophoresis.- 3.3.1 Sodium Dodecylsulphate-Polyacrylamide Gel Electrophoresis.- 3.3.2 Isoelectric Focusing.- 4 Lignins.- References.- Isolation and Analysis of Cell Wall Polymers from Olive Pulp.- 1 Introduction.- 2 Isolation of Cell Walls from Olive Pulp.- 2.1 Preparation and Use of Alcohol-Insoluble Residue (AIR) - General Considerations.- 2.2 Preparation of Cell Wall Material (CWM).- 2.2.1 Material Solubilized During the Preparation of CWM.- 3 Sequential Extraction of Cell Wall Polymers.- 3.1 General Considerations.- 3.2 Sequential Extraction.- 3.2.1 General Comments to the Method.- 4 Fractionation of the Extracted Cell Wall Polysaccharides.- 4.1 General Considerations.- 4.2 Graded Precipitation with Ethanol.- 4.3 Anion-Exchange Chromatography.- 4.3.1 Anion-Exchange Chromatography of Pectic Polysaccharides.- 4.3.2 Anion-Exchange Chromatography of Hemicellulosic Polysaccharides.- 4.4 Fractionation of Acidic Xylans.- 5 Chemical and Spectroscopic Analysis.- 5.1 Neutral Sugars.- 5.2 Uronic Acid.- 5.3 Hydroxyproline Estimation.- 5.4 Methylation Analysis.- 5.4.1 Hakomori Methylation.- 5.4.2 Ciucanu and Kerek Methylation.- 5.4.3 Carboxyl-Reduction of Methylated Polysaccharides.- 5.4.4 Comments on Results of Methylation Analysis.- 5.5 13C-NMR Studies on Cell Wall Polysaccharides.- 5.6 Fourier Transform Infrared Spectroscopy (FTIR).- 6 Concluding Remarks.- References.- Determination of Cell Wall Autolysis.- 1 Introduction.- 2 Factors Altering Cell Wall Isolation and Autolysis.- 2.1 Plant Material.- 2.2 Buffers.- 2.3 pH.- 2.4 Ionic Strength.- 3 Isolation of Active Cell Walls.- 3.1 Homogenization in Aqueous Medium.- 3.2 Filtration and Washes.- 4 Isolation of Inactive Cell Walls (Controls).- 4.1 Boiled in Hot Water.- 4.2 Boiled in Alcohol.- 4.3 Phenol-Acetic Acid-Water.- 4.4 Buffer Phenol at pH 7.- 4.5 Low Temperatures.- 5 Autolysis Incubations.- 5.1 Optimization of the Autolysis Reaction.- 5.2 General Procedure.- 6 Analysis of Autolysis Products.- 6.1 General Methods.- 6.2 Ethanol Precipitation.- 6.3 Gel Permeation Chromatography.- 6.4 Ion Exchange Chromatography.- 7 Conclusions.- References.- Cell Wall Porosity and Its Determination.- 1 Introduction.- 1.1 Biological Significance of Wall Porosity.- 1.2 Assumptions and Definitions.- 2 Microscopic Visualization of Wall Pores.- 3 Bulk Exclusion Techniques.- 3.1 Solute Exclusion.- 3.2 Long-Term-Exclusion of PEG (Polyethylenglycol).- 3.3 Gel Filtration.- 4 Tracer Techniques: Uptake of Molecules or Particles.- 4.1 Small Molecules.- 4.2 Macromolecules.- 4.3 Particles.- 5 Conclusions.- 5.1 Integration of Results from Different Methods.- 5.2 Pore Structure.- 5.3 Variations in Wall Porosity.- 5.4 Future Developments.- References.- Analysis of Chitin Biosynthesis.- 1 Introduction.- 2 Structure of Chitin.- 3 The Enzymatic Synthesis of Chitin.- 3.1 Assay of Chitin Synthase.- 3.2 Inhibition of Enzymatic Activity.- 4 Preparation of Chitin Synthase.- 5 Product Characterization of Chitin Synthase.- 5.1 Chemical Identification.- 5.2 Biophysical Identification of Chitin.- 5.2.1 Fourier Transform Infrared Spectroscopy.- 5.2.2 X-Ray Diffraction Analysis.- 5.2.3 Electron Microscopy and Electron Diffraction Analysis.- 6 Polymerization and Crystallization.- 7 Fungal Chitin Synthase Genes.- 8 Morphogenetic Roles of Chitin Synthases.- 9 Conclusions.- References.- Analysis of Plant-Substratum Adhesives.- 1 Introduction.- 1.1 Principles of Adhesion.- 1.2 Cell-Substratum Adhesion.- 1.2.1 Higher Plant-Substratum Adhesion.- 1.2.2 Adhesion of Fungal Phytopathogens to a Plant Substratum.- 1.2.3 Algal-Substratum Adhesion.- 2 Case Studies of Cell-Substratum Adhesion.- 2.1 Substrate Adhesion by the Fucus Zygote.- 2.2 Adhesion of Conidiospores of the Plant Pathogenic Fungus, Nectria haematococca.- 3 Methods for the Analysis of Cell-Substratum Adhesives.- 3.1 Adhesion Systems and Assays.- 3.2 Identification of Adhesive Components.- 3.2.1 Isolation of Adhesion Mutants.- 3.2.2 Correlation of Temporal/Spatial Development with Adhesion.- 3.2.3 Experimental Perturbation of Adhesion.- 3.3 Analysis of Adhesive Components.- 3.3.1 Extraction and Purification.- 3.3.2 Assays of Isolated Compounds for Adhesive Activity.- 4 Conclusions.- References.- Biochemical, Immunological and Molecular Analyses of Extensin.- 1 Introduction.- 2 Biochemical Characterization of Extensin.- 2.1 Preparation of Walls.- 2.2 Protein Purification.- 2.3 Biochemical Characterization.- 3 Immunological Detection of Extensin.- 3.1 In Vivo Localization of Extensin.- 3.2 Western Blots and Tissue Prints.- 4 Molecular Characterization of Extensin.- 4.1 Isolation of Genes Encoding Extensin.- 4.2 Extensin Expression Studies.- 4.3 Extensin Promoter Fusions with Reporter Genes.- 4.4 Generation of Extensin Mutants.- 5 Summary.- References.- Analysis of Pectin Structure by HPAEC-PAD.- 1 Introduction.- 2 Pectin HPLC Separations.- 2.1 GPC.- 2.2 Ion-Exchange and Ion-Pair RP.- 2.3 HPAEC-PAD.- 3 Pectin Analysis in Ripening Peach Fruit.- 3.1 Melting- and Nonmelting-Flesh Peaches.- 3.2 HPAEC-PAD System.- 3.3 Sample Preparation.- 3.4 HPAEC-PAD of Redskin and Suncling Peach Pectin.- 4 Conclusions and Future Directions.- References.- Characterization of Oligosaccharides Derived from Plant Cell Wall Polysaccharides by On-Line High-Performance Anion-Exchange Chromatography Thermospray Mass Spectrometry.- 1 Introduction.- 2 Methods.- 2.1 Apparatus.- 2.2 Isolation of Oligosaccharides from Plant Cell Wall Material.- 3 HPAEC in Oligosaccharide Analysis.- 4 General Experimental Considerations Related to HPAEC-MS.- 4.1 Desalting by AMMS.- 4.2 Ionization of Oligosaccharides.- 4.3 Data Interpretation.- 5 Application of HPAEC-MS in Oligosaccharide Characterization.- 6 Conclusions and Perspectives.- References.- Analysis of Pectin Methyl Esterases.- 1 Introduction.- 2 Estimation and Detection of PME Activities.- 2.1 Detection.- 2.2 Estimation.- 3 PME Localization.- 4 PME Extraction and Purification.- 5 PME Properties.- 5.1 Physicochemical Properties.- 5.2 Enzymic Properties.- 5.2.1 Action Pattern.- 5.2.2 Influence of pH on PME Activity.- 5.2.3 Influence of Cations.- 5.3 Structures.- 6 Roles of PMEs.- 6.1 Plant Pathogens.- 6.2 Fruit Maturation.- 6.3 Cell Elongation.- 7 Conclusions and Perspectives.- References.- Probing the Subunit Composition and Topology of Plasma Membrane-Bound (1,3)-?-Glucan (Callose) Synthases.- 1 Introduction.- 2 Membrane Isolation, Enzyme Assay, and Solubilization.- 2.1 Isolation of Crude Membrane Fractions.- 2.2 Plasma Membrane Isolation.- 2.3 Membranes of Defined Sidedness: Inside Out and Right-Side Out Vesicles.- 2.4 Callose Synthase Assay.- 2.5 Callose Synthase Solubilization.- 3 Callose Synthase Topology, Purification, and Subunit Composition.- 3.1 Polypeptide Depletion.- 3.2 Vectorial Proteolysis.- 3.3 Callose Synthase Purification.- 3.3.1 Glycerol Gradient Centrifugation.- 3.3.2 Product Entrapment.- 4 Biochemical Characterization of Integral Plant Plasma Membrane Proteins.- 4.1 Characterization of Disulfide-Linked Aggregate Formation.- 4.2 Recovery of Hydrophobic Plant-Derived Membrane Proteins for Sequencing and Antibody Production.- 4.3 Antibody Characterization.- 4.3.1 Immunoblotting.- 4.3.2 Affinity Purification of Anti-PMIP27.- 5 Summary.- References.