Plant Cell Wall Analysis

ISBN-13: 9783642646447 / Angielski / Miękka / 2011 / 205 str.

Hans F. Linskens; John F. Jackson

Opis

This book brings together techniques dealing with those parts of plant cells which have often been thought of as being extremely difficult to analyse. Cell wall porosity adhesives, proteins, chitin, lignin, oligosaccharides, pectins and other polymers are all the subject of analytical procedures in this volume. Wherever possible, the methods stand alone with little or no necessity to consult other works to carry out the methods put forward. Each chapter has been written by experts in the field using the most up-to-date analysis techniques.

Spis treści

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.