Table of Contents

Chapter 1: History & Background
1.1 Temperature Stress
1.2 The Heat Shock Response
1.3 Heat Shock Protein Families
1.4 Heat Shock Genes
1.5 Heat Shock Transcription Factors
1.6 Regulation of the Heat Shock Response
1.7 The Arabidopsis HSP & HSF Families
1.8 Low Temperature Stress in Plants
1.9 Low Temperature Stress & HSPs
1.10 Temperature Stress in Alfalfa (Medicago sativa L.)
1.11 Research Hypothesis

Chapter 2: Isolation, Localization and Sequence Analysis of an Alfalfa HSF
2.1 Introduction
2.2 Material & Methods
2.2.1 32P Probe Preparation
2.2.2 ? Phage cDNA Library Construction, Preparation & Titer Estimation
2.2.3 ? Phage cDNA Library Screening
2.2.3.1 Plating the Library & Plaque Lifts
2.2.3.2 Plaque Hybridization
2.2.3.3 Positive Plaque Identification
2.2.4 Sequence Analysis
2.2.5 Isolation of Genomic DNA
2.2.6 Genomic Library Construction
2.2.7 Genomic Library Screening
2.2.8 Digoxigenin (DIG) Probe Preparation
2.2.9 Southern Blot Hybridization
2.2.10 Fluorescent In Situ Hybridization
2.2.11 RT-PCR
2.3 Results
2.3.1 cDNA Sequence
2.3.1.1 5’ UTR
2.3.2 Amino Acid Sequence
2.3.3 Genomic Sequence
2.3.4 Genomic Localization
2.4 Discussion
2.5 Conclusion

Chapter 3: Endogenous HSF Activity in Alfalfa
3.1 Introduction
3.2 Materials & Methods
3.2.1 Tobacco Protoplast Expression
3.2.1.1 Vector Construct Construction
3.2.1.2 Protoplast Transformation, Expression & Evaluation
3.2.2 Expression of Excised Apical Shoots
3.2.3 Germplasm
3.2.4 Diurnal Experiment
3.2.5 Chamber Experiment - In vivo Expression
3.2.6 Field Experiment
3.2.7 RNA Isolation
3.2.8 RNA Quantification
3.2.9 RNA Quality
3.2.10 DIG Probe Preparation
3.2.11 Northern Blot Hybridization
3.2.12 Preparation of RNA Macroarrays
3.2.13 Screening of RNA Macroarrays
3.2.14 Statistical Analysis
3.3 Results
3.3.1 Activity of MsHSFA4 in Tobacco Protoplasts
3.3.2 Northern Blot of MsHSFA4 Temperature Expression
3.3.3 HSF Diurnal Effect
3.3.4 Chamber Experiment - Heat and Cold Stress
3.3.4.1 Heat Stress
3.3.4.2 Cold Stress
3.3.5 Field Experiment - Fall 2001
3.4 Discussion
3.5 Conclusion

Chapter 4: Expression of Heat Shock Genes and HSPs
4.1 Introduction
4.2 Materials & Methods
4.2.1 DIG Probe Preparation
4.2.2 Rescreening of RNA Macroarrays
4.2.3 Protein Tissue Preparation
4.2.4 Protein Extraction
4.2.5 Protein Quantification
4.2.6 Western Blot Immuno-detection
4.2.7 Preparation of Protein Macroarrays
4.2.8 Screening of Protein Macroarrays
4.2.9 Statistical Analysis
4.3 Results
4.3.1 Transcript Expression of HSP 18 mRNA, HSP 86 mRNA and Protein Accumulations of Low Molecular Weight HSPs Under Heat Stress 41°C
4.3.1.1 Chamber Experiment - HSP 18 mRNA
4.3.1.2 Chamber Experiment - HSP 86 mRNA
4.3.1.3 Laboratory and Chamber Experiment - lmwHSPs
4.3.2 Transcript Expression of HSP 18 mRNA, HSP 86 mRNA and Protein Accumulations of Low Molecular Weight HSPs Under Cold Stress at 4°C
4.3.2.1 Chamber Experiment - HSP 18 mRNA
4.3.2.2 Chamber Experiment - HSP 86 mRNA
4.3.2.3 Chamber Experiment - lmwHSPs
4.3.3 Transcript Expression of HSP 18 mRNA, HSP 86 mRNA and Protein Accumulations of Low Molecular Weight HSPs Under Field Fall Conditions in 2001
4.3.3.1 HSP 18 mRNA - Root and Bud Expression
4.3.3.2 HSP 86 mRNA - Root and Bud Expression
4.3.3.3 Low Molecular Weight HSPs - Root and Bud Expression
4.4 Discussion
4.5 Conclusion

Chapter 5: Engineering an HSF in Alfalfa
5.1 Introduction
5.2 Materials and Methods
5.2.1 Binary Vector Construction
5.2.2 Agrobacterium Mediated Transformation
5.2.2.1 Transformation
5.2.2.2 Tissue Culture & Selection
5.2.3 Conformation of Transgene Insertion
5.2.3.1 PCR
5.2.3.2 Southern Blot Hybridization
5.2.3.3 Northern Blot Hybridization
5.2.3.4 RT-PCR
5.2.4 Testcross and Transgene Inheritance
5.2.5 Field Experiment
5.2.5.1 To Primary Transgenics
5.2.5.2 T1 Transgenic Testcross Progeny
5.3 Results
5.3.1 Transgene Integration
5.3.1.1 DNA Analysis
5.3.1.2 RNA Analysis
5.3.1.3 Transgene Segregation
5.3.2 Field Experiment
5.3.2.1 To Primary Transgenics
5.3.2.2 F1 Progeny Transgenics
5.4 Discussion
5.5 Conclusion

Chapter 6: General Discussion & Conclusions
6.1 The Heat Shock Response in Alfalfa under Heat Stress
6.2 The Heat Shock Response in Alfalfa under Cold Stress
6.3 The Heat Shock Response in Alfalfa during the Fall
6.4 The Diurnal Effect of Alfalfa HSF
6.5 Differences in HS Gene Expression among Alfalfa Cultivars
6.6 The Connection between Heat and Cold Stress
6.7 The Utility of Engineering HSF to Protect Against Low Temperature Stress

Literature Cited

Appendices
Appendix A: List of PCR Primers
Appendix B Solution Recipes
Appendix C: Species Phylogeny
Appendix D: MsHSFA4 cDNA Sequence
Appendix E: MsHSFA4 Genomic Sequence
Appendix F: Fertilizer Nutrient Composition
Appendix G: DIG Probe Conformation of HSP 18 and HSP 86
Appendix H: Example of PCR Analysis of Transgenic Progeny
Appendix I: Design of the Chamber Experiment
Appendix J: Design of the Cultivar Field Experiment
Appendix K: Design of the Transgenic Experimental Blocks
Appendix L: Design of the Transgenic Field Experiment
Appendix M: Total Protein Accumulation during the Fall Field Experiment