Plant cell biology : a practical approach / 2nd ed.
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作 者：edited by Chris Hawes and B鈋atrice Satiat-Jeunemaitre.
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ISBN：9780199638659
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简介
Summary:
Publisher Summary 1
Surveys techniques for studying the location and function of proteins within a cell, with an emphasis on microscopy. The 13 chapters introduce fluorescent probes, cytometry, microinjection, micromanipulation, the isolation of cytoplasmic organelles, and classical histochemistry. The second edition adds chapters on the expression of heterologous proteins in protoplasts and the green fluorescent protein. Annotation c. Book News, Inc., Portland, OR (booknews.com)
Publisher Summary 2
With the 'post genomics' era comes an increasing demand for the techniques of cell biology, critical to interpreting the function and location of the cell's myriad proteins and macromolecules. In response, this second edition of Plant Cell Biologybalances established techniques, including classical histochemistry and electron microscopy, with new developments in the field. The book covers a substantial range of methods for working on living cells, including the application of fluorescent probes, cytometry, expression systems, the use of green fluorescent protein, micromanipulation and electrophysiological techniques. Also featured are chapters on macromolecular location procedures involving immunocytochemistry and in situhybridisation, and the book concludes with a range of biochemical techniques for the isolation of cytoplasmic organelles. The book provides advanced students, postgraduates and researchers in the plant sciences with an invaluably comprehensive guide to the ever-growing field of plant cell biology.
目录
List of protocols p. xv
Abbreviations p. xix
Introduction to optical microscopy for plant cell biology p. 1
Introduction p. 1
Explanation of terms p. 4
Recording images p. 7
Image resolution p. 7
Film recording p. 8
Electronic cameras p. 9
Microscope imaging modes p. 10
Bright field imaging p. 10
Phase contrast p. 12
Differential interference contrast (Nomarski) p. 14
Dark field p. 16
Epifluorescence and reflected light microscopy p. 17
Confocal and 3D microscopy p. 19
The problem of out-of-focus light p. 19
The confocal principle: explanation by ray optics p. 20
Practical confocal microscopes p. 21
Imaging and the point spread function p. 23
Deconvolution p. 24
Two photon imaging p. 26
Comparison of conventional, wide-field fluorescence imaging with confocal fluorescence imaging p. 27
Noise and resolution p. 27
When should confocal microscopy be used? p. 29
Objective lenses for confocal imaging p. 30
Specimen preparation for confocal imaging p. 30
References p. 33
Fluorescent probes for living plant cells p. 35
Introduction p. 35
Selecting probes with high brightness p. 35
Spectral considerations p. 37
Fluorescence lifetime imaging microscopy (FLIM) p. 38
Fluorescence polarization anisotropy p. 38
Fluorescence resonance energy transfer (FRET) p. 38
Photobleaching and fluorescence redistribution after photobleaching (FRAP) p. 39
Optimization of fluorescent systems for live cell imaging p. 40
Selection of the excitation wavelength p. 41
The dichroic mirror p. 41
Selection of the emission wavelength p. 41
Choice of measurement system p. 42
Securing the specimen for microscopy p. 43
Perfusion systems p. 45
Loading strategies for plant cells p. 46
Extracellular and permeant intracellular dyes p. 46
Ester loading p. 47
Low pH loading p. 48
Cutinase pre-treatment and low pH loading p. 49
Electroporation p. 50
Loading via detergent permeabilization p. 50
Loading tissues with phloem-mobile probes p. 50
Intracellular dye concentrations, viability, and toxicity p. 53
Selection and use of fluorescent probes p. 54
Vital stains p. 55
Mortal stains p. 56
Cell permeant nuclear stains p. 57
Chloroplasts p. 57
Mitochondria p. 58
Vacuoles p. 60
Endoplasmic reticulum p. 62
Golgi p. 62
Cytoskeleton p. 63
The plasma membrane and endocytosis p. 64
The cell wall p. 65
Physiological probes p. 66
Calcium p. 66
Measurement of apoplastic, cytoplasmic, and vacuolar pH p. 72
Potassium p. 76
Aluminium p. 76
Measurement of cytoplasmic glutathione levels p. 77
Reactive oxygen species p. 77
Data analysis p. 78
Attenuation correction for optical sections deep into tissues p. 80
Acknowledgements p. 81
References p. 81
Flow cytometry p. 85
Introduction p. 85
Cytometry demonstrated through cell cycle analyses p. 86
How to understand monoparametric DNA histograms p. 87
Developing multiparametric DNA histograms and immunofluorescence p. 90
Extracting intact plant nuclei p. 91
Which DNA fluorochrome is appropriate? p. 94
Running and reading the cytometer p. 94
BrdU incorporation to identify DNA synthesis by fluorescence quenching p. 95
The particular application of genome size calculation and 'DNA ploidy' p. 99
Terminology p. 99
Internal or external standards p. 99
Calculating base composition p. 100
Sorting of protoplasts and cellular organelles p. 101
Tests for cell viability during functional assays p. 104
Conclusion p. 104
Acknowledgements p. 104
References p. 105
Transient expression, a tool to address questions in plant cell biology p. 107
Introduction p. 107
Current methods of transient expression p. 108
Naked DNA transfer p. 108
Biological vectors p. 108
Application of transient expression p. 109
Promoter analysis p. 109
Cell biology and biochemistry p. 110
Practical considerations for cell biologists p. 111
Naked DNA transfer p. 112
Measurement of protein secretion and cell retention p. 116
Large scale transient expression for cell fractionation p. 118
Specialized applications p. 122
Conclusions p. 124
References p. 124
The green fluorescent protein (GFP) as reporter in plant cells p. 127
Introduction p. 127
The green fluorescent protein p. 127
Structure p. 127
GFP variants p. 128
GFP as a reporter for gene expression p. 128
GFP as a reporter for protein location p. 129
Cytoplasm and nucleus p. 129
Chloroplasts and mitochondria p. 130
Secretory pathway p. 130
Viral proteins p. 131
Transformation methods p. 131
PEG-mediated transient expression in protoplasts p. 131
Agrobacterium-mediated transient expression in planta p. 134
Virus-mediated transient expression p. 136
Visualization and microscopy of GFP p. 140
Future perspectives p. 141
References p. 141
Microinjection p. 143
Introduction p. 143
Equipment p. 143
Environment and injection-table p. 143
Microscope p. 144
Objectives p. 144
Glass capillaries p. 145
Tip puller p. 145
Micromanipulator p. 146
Injection techniques p. 146
Iontophoresis p. 146
Pressure injection p. 146
The galinstan expansion femtosyringe (GEF) p. 147
Cell types p. 149
Epidermal cells p. 149
Guard cells and trichomes p. 150
Mesophyll cells p. 150
Ground parenchyma cells p. 151
Sieve elements and companion cells p. 151
Algae p. 152
Bacteria and organelles p. 153
Plant tissue cultures p. 153
Material suitable for injection p. 154
Fluorochromes p. 154
Dextran conjugates p. 154
Proteins and antibodies p. 155
Nucleic acids p. 155
Tips to make life easier p. 155
The chuck p. 155
Syringe to delete air bubbles p. 156
Flexible fused silica capillaries p. 156
Petri dishes p. 157
References p. 157
Micromanipulation by laser microbeam and optical tweezers p. 159
Introduction p. 159
What are laser microtools? p. 159
Physical background p. 160
Generating extreme heat p. 160
Why can light be used to move microscopic objects? p. 160
How to build laser microtools p. 161
The choice of lasers p. 161
Building a laser microbeam or optical tweezers p. 162
Applications of laser microbeams in plant biology p. 163
Laser-induced microinjection p. 163
Ablation to study cell fate during plant development p. 164
Protoplast fusion p. 165
Preparation of cell membranes from root hairs p. 165
Applications of optical tweezers to plant biology p. 166
Capturing subcellular organelles for inspection p. 166
Simulating microgravity p. 167
Conclusion p. 168
References p. 168
Electrophysiological methods: monitoring exo- and endocytosis in real time p. 171
Introduction p. 171
Theoretical background p. 171
The membrane is equivalent to a capacitor p. 171
A cell as an equivalent circuit p. 172
Techniques for the measurement of membrane capacitance p. 172
Square-wave stimulation: time-domain technique p. 173
Saw-tooth stimulation p. 174
Capacitance cancellation p. 174
Sinusoidal excitation p. 174
Capacitance measurements as an assay for exo- and endocytosis: practical considerations p. 175
What kind of cells can be examined? p. 176
Estimation of the specific capacitance p. 176
Recording of single fusion and fission events p. 176
What kind of information can be extracted from the measurements? p. 179
Macroscopic measurement of membrane capacitance p. 181
Acknowledgements p. 186
References p. 187
Plant histology p. 189
Introduction p. 189
Conventional chemical fixation methods p. 189
Conventional embedding methods and sectioning p. 191
Embedding in a matrix p. 192
Frozen sections p. 194
Conventional staining methods p. 195
General tissue stains p. 195
Cell wall stains p. 197
Carbohydrate and starch stains p. 200
Lipid stains p. 201
Nucleic acid stains p. 202
Miscellaneous staining methods p. 203
References p. 206
Immunocytochemistry for light microscopy p. 207
Introduction p. 207
Principles and use of immunocytochemistry p. 208
Direct and indirect immunostaining p. 208
The antibody-antigen complex p. 208
Whole molecules or fragments p. 209
Polyclonal and monoclonal antibodies p. 211
When to perform in situ immunoreaction p. 214
Antibodies to epitope tags p. 215
Basic methods for immunostaining p. 215
Preparing plant material p. 216
Attaching material to slides and coverslips p. 220
Accessing epitopes in cells p. 220
Counterstaining and mounting p. 230
Interpreting the immunostaining pattern p. 230
Multiple staining p. 231
Multiple immunostaining p. 231
Combining immunostaining with other affinity techniques p. 231
Conclusion p. 232
References p. 232
Electron microscopy p. 235
Introduction p. 235
Transmission electrom microscopy p. 235
Conventional methods p. 235
Low temperature methods p. 250
Rotary shadowing of proteins p. 257
Scanning electron microscopy p. 258
Ambient temperature SEM p. 258
Low temperature SEM p. 262
Immuno-SEM p. 263
Acknowledgements p. 263
References p. 264
In situ hybridization p. 267
Introduction p. 267
Applications of in situ hybridization p. 269
DNA:DNA in situ hybridization p. 269
RNA:RNA in situ hybridization p. 269
Background to the methods p. 269
Chromosome preparation for DNA:DNA in situ hybridization p. 270
Animal material p. 270
Plant material p. 271
Material preparation for RNA:RNA in situ hybridization p. 273
Specimen preparation p. 273
Controls p. 275
Labelling the nucleic acids p. 276
DNA labelling p. 276
Checking probe incorporation p. 282
Material pre-treatment p. 283
Pre-treatment for DNA:DNA in situ hybridization p. 283
Pre-treatment for RNA:RNA in situ hybridization p. 284
In situ hybridization reaction p. 285
DNA:DNA in situ hybridization p. 285
RNA:RNA in situ hybridization p. 286
Post-hybridization washes p. 287
Washes for DNA:DNA in situ hybridization p. 288
Washes for RNA:RNA in situ hybridization p. 288
Probe detection and visualization p. 289
Visualization p. 290
Epifluorescence microscopy p. 290
Image capture p. 291
Image manipulation p. 292
References p. 292
Organelle isolation p. 295
Introduction p. 295
General methodology p. 295
Homogenizing media p. 295
Methods of homogenization p. 298
Methods of organelle separation p. 299
Isolation of chloroplasts p. 300
Isolation of mitochondria p. 300
Isolation of nuclei p. 301
Isolation of microbodies p. 303
Isolation of plasma membrane p. 304
Isolation of tonoplast p. 306
Isolation of endoplasmic reticulum p. 308
Isolation of Golgi apparatus p. 310
Isolation of transport vesicles p. 312
Assays for marker enzymes p. 315
Antibodies for organelle recognition p. 319
References p. 320
List of suppliers p. 325
Index p. 333
Abbreviations p. xix
Introduction to optical microscopy for plant cell biology p. 1
Introduction p. 1
Explanation of terms p. 4
Recording images p. 7
Image resolution p. 7
Film recording p. 8
Electronic cameras p. 9
Microscope imaging modes p. 10
Bright field imaging p. 10
Phase contrast p. 12
Differential interference contrast (Nomarski) p. 14
Dark field p. 16
Epifluorescence and reflected light microscopy p. 17
Confocal and 3D microscopy p. 19
The problem of out-of-focus light p. 19
The confocal principle: explanation by ray optics p. 20
Practical confocal microscopes p. 21
Imaging and the point spread function p. 23
Deconvolution p. 24
Two photon imaging p. 26
Comparison of conventional, wide-field fluorescence imaging with confocal fluorescence imaging p. 27
Noise and resolution p. 27
When should confocal microscopy be used? p. 29
Objective lenses for confocal imaging p. 30
Specimen preparation for confocal imaging p. 30
References p. 33
Fluorescent probes for living plant cells p. 35
Introduction p. 35
Selecting probes with high brightness p. 35
Spectral considerations p. 37
Fluorescence lifetime imaging microscopy (FLIM) p. 38
Fluorescence polarization anisotropy p. 38
Fluorescence resonance energy transfer (FRET) p. 38
Photobleaching and fluorescence redistribution after photobleaching (FRAP) p. 39
Optimization of fluorescent systems for live cell imaging p. 40
Selection of the excitation wavelength p. 41
The dichroic mirror p. 41
Selection of the emission wavelength p. 41
Choice of measurement system p. 42
Securing the specimen for microscopy p. 43
Perfusion systems p. 45
Loading strategies for plant cells p. 46
Extracellular and permeant intracellular dyes p. 46
Ester loading p. 47
Low pH loading p. 48
Cutinase pre-treatment and low pH loading p. 49
Electroporation p. 50
Loading via detergent permeabilization p. 50
Loading tissues with phloem-mobile probes p. 50
Intracellular dye concentrations, viability, and toxicity p. 53
Selection and use of fluorescent probes p. 54
Vital stains p. 55
Mortal stains p. 56
Cell permeant nuclear stains p. 57
Chloroplasts p. 57
Mitochondria p. 58
Vacuoles p. 60
Endoplasmic reticulum p. 62
Golgi p. 62
Cytoskeleton p. 63
The plasma membrane and endocytosis p. 64
The cell wall p. 65
Physiological probes p. 66
Calcium p. 66
Measurement of apoplastic, cytoplasmic, and vacuolar pH p. 72
Potassium p. 76
Aluminium p. 76
Measurement of cytoplasmic glutathione levels p. 77
Reactive oxygen species p. 77
Data analysis p. 78
Attenuation correction for optical sections deep into tissues p. 80
Acknowledgements p. 81
References p. 81
Flow cytometry p. 85
Introduction p. 85
Cytometry demonstrated through cell cycle analyses p. 86
How to understand monoparametric DNA histograms p. 87
Developing multiparametric DNA histograms and immunofluorescence p. 90
Extracting intact plant nuclei p. 91
Which DNA fluorochrome is appropriate? p. 94
Running and reading the cytometer p. 94
BrdU incorporation to identify DNA synthesis by fluorescence quenching p. 95
The particular application of genome size calculation and 'DNA ploidy' p. 99
Terminology p. 99
Internal or external standards p. 99
Calculating base composition p. 100
Sorting of protoplasts and cellular organelles p. 101
Tests for cell viability during functional assays p. 104
Conclusion p. 104
Acknowledgements p. 104
References p. 105
Transient expression, a tool to address questions in plant cell biology p. 107
Introduction p. 107
Current methods of transient expression p. 108
Naked DNA transfer p. 108
Biological vectors p. 108
Application of transient expression p. 109
Promoter analysis p. 109
Cell biology and biochemistry p. 110
Practical considerations for cell biologists p. 111
Naked DNA transfer p. 112
Measurement of protein secretion and cell retention p. 116
Large scale transient expression for cell fractionation p. 118
Specialized applications p. 122
Conclusions p. 124
References p. 124
The green fluorescent protein (GFP) as reporter in plant cells p. 127
Introduction p. 127
The green fluorescent protein p. 127
Structure p. 127
GFP variants p. 128
GFP as a reporter for gene expression p. 128
GFP as a reporter for protein location p. 129
Cytoplasm and nucleus p. 129
Chloroplasts and mitochondria p. 130
Secretory pathway p. 130
Viral proteins p. 131
Transformation methods p. 131
PEG-mediated transient expression in protoplasts p. 131
Agrobacterium-mediated transient expression in planta p. 134
Virus-mediated transient expression p. 136
Visualization and microscopy of GFP p. 140
Future perspectives p. 141
References p. 141
Microinjection p. 143
Introduction p. 143
Equipment p. 143
Environment and injection-table p. 143
Microscope p. 144
Objectives p. 144
Glass capillaries p. 145
Tip puller p. 145
Micromanipulator p. 146
Injection techniques p. 146
Iontophoresis p. 146
Pressure injection p. 146
The galinstan expansion femtosyringe (GEF) p. 147
Cell types p. 149
Epidermal cells p. 149
Guard cells and trichomes p. 150
Mesophyll cells p. 150
Ground parenchyma cells p. 151
Sieve elements and companion cells p. 151
Algae p. 152
Bacteria and organelles p. 153
Plant tissue cultures p. 153
Material suitable for injection p. 154
Fluorochromes p. 154
Dextran conjugates p. 154
Proteins and antibodies p. 155
Nucleic acids p. 155
Tips to make life easier p. 155
The chuck p. 155
Syringe to delete air bubbles p. 156
Flexible fused silica capillaries p. 156
Petri dishes p. 157
References p. 157
Micromanipulation by laser microbeam and optical tweezers p. 159
Introduction p. 159
What are laser microtools? p. 159
Physical background p. 160
Generating extreme heat p. 160
Why can light be used to move microscopic objects? p. 160
How to build laser microtools p. 161
The choice of lasers p. 161
Building a laser microbeam or optical tweezers p. 162
Applications of laser microbeams in plant biology p. 163
Laser-induced microinjection p. 163
Ablation to study cell fate during plant development p. 164
Protoplast fusion p. 165
Preparation of cell membranes from root hairs p. 165
Applications of optical tweezers to plant biology p. 166
Capturing subcellular organelles for inspection p. 166
Simulating microgravity p. 167
Conclusion p. 168
References p. 168
Electrophysiological methods: monitoring exo- and endocytosis in real time p. 171
Introduction p. 171
Theoretical background p. 171
The membrane is equivalent to a capacitor p. 171
A cell as an equivalent circuit p. 172
Techniques for the measurement of membrane capacitance p. 172
Square-wave stimulation: time-domain technique p. 173
Saw-tooth stimulation p. 174
Capacitance cancellation p. 174
Sinusoidal excitation p. 174
Capacitance measurements as an assay for exo- and endocytosis: practical considerations p. 175
What kind of cells can be examined? p. 176
Estimation of the specific capacitance p. 176
Recording of single fusion and fission events p. 176
What kind of information can be extracted from the measurements? p. 179
Macroscopic measurement of membrane capacitance p. 181
Acknowledgements p. 186
References p. 187
Plant histology p. 189
Introduction p. 189
Conventional chemical fixation methods p. 189
Conventional embedding methods and sectioning p. 191
Embedding in a matrix p. 192
Frozen sections p. 194
Conventional staining methods p. 195
General tissue stains p. 195
Cell wall stains p. 197
Carbohydrate and starch stains p. 200
Lipid stains p. 201
Nucleic acid stains p. 202
Miscellaneous staining methods p. 203
References p. 206
Immunocytochemistry for light microscopy p. 207
Introduction p. 207
Principles and use of immunocytochemistry p. 208
Direct and indirect immunostaining p. 208
The antibody-antigen complex p. 208
Whole molecules or fragments p. 209
Polyclonal and monoclonal antibodies p. 211
When to perform in situ immunoreaction p. 214
Antibodies to epitope tags p. 215
Basic methods for immunostaining p. 215
Preparing plant material p. 216
Attaching material to slides and coverslips p. 220
Accessing epitopes in cells p. 220
Counterstaining and mounting p. 230
Interpreting the immunostaining pattern p. 230
Multiple staining p. 231
Multiple immunostaining p. 231
Combining immunostaining with other affinity techniques p. 231
Conclusion p. 232
References p. 232
Electron microscopy p. 235
Introduction p. 235
Transmission electrom microscopy p. 235
Conventional methods p. 235
Low temperature methods p. 250
Rotary shadowing of proteins p. 257
Scanning electron microscopy p. 258
Ambient temperature SEM p. 258
Low temperature SEM p. 262
Immuno-SEM p. 263
Acknowledgements p. 263
References p. 264
In situ hybridization p. 267
Introduction p. 267
Applications of in situ hybridization p. 269
DNA:DNA in situ hybridization p. 269
RNA:RNA in situ hybridization p. 269
Background to the methods p. 269
Chromosome preparation for DNA:DNA in situ hybridization p. 270
Animal material p. 270
Plant material p. 271
Material preparation for RNA:RNA in situ hybridization p. 273
Specimen preparation p. 273
Controls p. 275
Labelling the nucleic acids p. 276
DNA labelling p. 276
Checking probe incorporation p. 282
Material pre-treatment p. 283
Pre-treatment for DNA:DNA in situ hybridization p. 283
Pre-treatment for RNA:RNA in situ hybridization p. 284
In situ hybridization reaction p. 285
DNA:DNA in situ hybridization p. 285
RNA:RNA in situ hybridization p. 286
Post-hybridization washes p. 287
Washes for DNA:DNA in situ hybridization p. 288
Washes for RNA:RNA in situ hybridization p. 288
Probe detection and visualization p. 289
Visualization p. 290
Epifluorescence microscopy p. 290
Image capture p. 291
Image manipulation p. 292
References p. 292
Organelle isolation p. 295
Introduction p. 295
General methodology p. 295
Homogenizing media p. 295
Methods of homogenization p. 298
Methods of organelle separation p. 299
Isolation of chloroplasts p. 300
Isolation of mitochondria p. 300
Isolation of nuclei p. 301
Isolation of microbodies p. 303
Isolation of plasma membrane p. 304
Isolation of tonoplast p. 306
Isolation of endoplasmic reticulum p. 308
Isolation of Golgi apparatus p. 310
Isolation of transport vesicles p. 312
Assays for marker enzymes p. 315
Antibodies for organelle recognition p. 319
References p. 320
List of suppliers p. 325
Index p. 333
Plant cell biology : a practical approach / 2nd ed.
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