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MOLECULAR AND GENETIC STUDIES OF IRON HOMEOSTASIS IN ARABIDOPSIS ___________________________________________ A Dissertation Presented to the Faculty of the Graduate School University of Missouri-Columbia ___________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy ___________________________________________ by ALBERTO MAURER Dr. Elizabeth E. Rogers Dissertation Supervisor MAY 2006 © Copyright by All Rights Reserved The undersigned, appointed by the Dean of the Graduate School, have examined the dissertation entitled MOLECULAR AND GENETIC STUDIES OF IRON HOMEOSTASIS IN ARABIDOPSIS Presented by ALBERTO MAURER A candidate for the degree of Doctor of Philosophy And hereby certify that in their opinion it is worthy of acceptance ______________________________ Dr. Elizabeth E. Rogers ______________________________ Dr. Michael Petris ______________________________ Dr. John C. Walker ______________________________ Dr. Walter Gassmann ______________________________ Dr. Joseph C. Polacco ACKNOWLEDGEMENTS First of all, I would like to thank my advisor, Dr. Elizabeth E. Rogers, for giving me the opportunity to complete my Ph.D. degree in her lab. I was only able to finish this thesis thanks to her patience and guidance. Second, I would like to thank all the people and organizations that were able to support my studies in the form of assistantships: The Genetics Area Program and its former director Dr. Donald Riddle for my first-year assistantship; Dr. Douglass Randall and the Interdisciplinary Plant Group for the support given to me in the form of a 3-year assistantship; Dr. Elizabeth E. Rogers who supported my studies for 2 years; and to the Monsanto Corporation, who awarded me a senior grad-student assistantship. Thanks to all of you I was able to complete my degree. I will be forever thankful to Dr. Donald Riddle, Dr. Joseph Polacco, and Dr. Kim Won-Seok for their enormous support while confronting difficult times in the middle of my degree. Many thanks to the past and present members of my Graduate Committee: you were always supportive and ready with suggestions and help for my research and graduate career: Dr. Michael Petris, Dr. John C. Walker, Dr. Walter Gassmann, Dr. David Eide, and especially Dr. Joseph Polacco. I need to thank my lab mates Mr. Timothy Durrett and Dr. Dirk Charlson. They were next to me in the trenches of our daily battle for good results and sound science. All their advice, suggestions, and help were primordial in the research that led to this thesis and my degree. I would like to include here all the rotation students, undergraduates, and ii visiting scholars, that in one way or another helped me complete experiments and projects: Lorena DaPonte, Timothy Pouland, Juan Gutierrez, Kareema Smith, and Sandhya Tondhapu. Your help was more valuable than you could ever imagine. A project as long and arduous as a Ph.D. degree in Genetics would have been impossible for me to attain without the unconditional support of my friends Miguel and Alicia Rodriguez, Moises Cortes-Cruz, Jason Meyer, Thomas Campbell, Juan Jose Gutierrez, Anna Glazkova, Carine Tannous, and many others that if mentioned, would fill many pages like this one. Thanks to you all. Finally, the most important thanks of all: to my family. Thanks for your encouragement, support, and patience. This degree is dedicated to you. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………ii LIST OF TABLES…………………………………………………………..v LIST OF FIGURES……………………………...…………………..……...vi ABSTRACT………………………………………………………………..vii CHAPTER 1. INTRODUCTION AND LITERATURE REVIEW……..1 CHAPTER 2. FUNCTIONAL GENOMIC ANALYSIS OF IRON HOMEOSTASIS IN ARABIDOPSIS THALIANA……...26 CHAPTER 3. INITIAL CHARACTERIZATION OF FOUR BHLH TRANSCRIPTION FACTORS UP- REGULATED IN RESPONSE TO IRON- DEFICIENCY CONDITIONS IN ARABIDOPSIS THALIANA……………….…………………………….....79 CHAPTER 4. DEFECTS IN CHLOROPLAST PURINE BIOSYNTHESIS ALTER METAL HOMEOSTASIS IN ARABIDOPSIS THALIANA………………………...135 VITA…...………………………………………………………….……...164 iv LIST OF TABLES Table Page Table 2.1. Affymetrix microarray results showing iron-regulated genes in shoots and roots of Arabidopsis thaliana Col-0 plants……...……..….56 Table 2.2. Affymetrix dataset from iron-sufficient and –deficient WT shoots or roots……………………………………………………….59 Table 2.3. Individual T-DNA lines ordered from ABRC for each of the selected genes………………………………………………….….62 Table 2.4. Results of the Affymetrix microarray for selected genes………………….63 Table 2.5. Elemental analysis of selected T-DNA insertion lines compared with Arabidopsis thalinana Col-0……………………………64 Table 2.6. 29 common sequences to all three selected P450s and four selection criteria to be selected for RNAi…………………………..65 Table 3.1. Transcript levels for genes At5g04150 and At3g56980…………….……107 Table 3.2. Identity in amino acid sequences between the four TFs……………….....107 Table 3.3. Gauntlet conditions used in the T-DNA and over-expressor lines……….108 Table 3.4. Elemental analysis of the TFs T-DNA lines…………………………...…109 Table 3.5. Expression of three iron-regulated genes in roots of iron- deficient WT and bHLH101 T-DNA plants……………………………110 Table 3.6. bHLH101 T-DNA Affymetrix microarray analysis………...……………111 Table 4.1. Average leaf elemental content of wild-type (WT) and SP46 Arabidopsis plants…………………………………………….….147 Table 4.2. Elemental analysis in ppm/gram dry weight of Arabidopsis plants grown in liquid media with or without 10mM inosine…………..147 v LIST OF FIGURES Figure Page Figure 2.1. Comparison of transcript levels as detected for genes in the microarray or in RNA blots………………………………………….35 Figure 2.2. Illustrations representing the arrangement of introns, exons, and T-DNA insertions in eight selected genes…………………………...42 Figure 2.3. Homology between three P450 genes…………………………………...…46 Figure 3.1. ClustalW tree of 20 closely–related bHLH transcription factors………………………………………………………………...…..84 Figure 3.2. ClustalW aligment of four closely related bHLH transcription factors……………...….………………………………..….85 Figure 3.3. Timeline of expression of selected bHLH transcription factors under iron-deficiency and control conditions in shoots and roots……………..………………………………………...….87 Figure 3.4. Diagrams of the four transcription factors and their T-DNA insertions…...………………………………………………….……...….89 Figure 3.5. Staining of bHLH101::GUS transgenic Arabidopsis plants……………….94 Figure 3.6. Diagram of the constructs used in our research……………………………95 Figure 3.7. bHLH101 transcript levels in the over-expressor transgenic lines…......….97 Figure 4.1. Phenotype of rosette leaves and 4-week-old plants………………………140 Figure 4.2. Time course of ferric-chelate reductase activity…………………...……..141 Figure 4.3. Map-based cloning of the SP46 locus.…………………………...….....…149 Figure 4.4. Amino acid sequence analysis of the AtATase family.………............…..150 vi MOLECULAR AND GENETIC STUDIES OF IRON HOMEOSTASIS IN ARABIDOPSIS Alberto Maurer Dr. Elizabeth E. Rogers, Dissertation Supervisor ABSTRACT Iron is an essential element for plant survival. However, in excess, it is deleterious to the organism, thus iron concentration is tightly regulated in planta. The first point in the iron regulation system is acquisition, and plants have evolved two different strategies to obtain it from the soil. Dicots and non-graminaceous monocots use Strategy I to obtain iron from soil, whereas grasses use a chelation-based mechanism termed Strategy II (Marschner, 1995 and see Chapter 1). In the present thesis, we describe two different approaches to increase our understanding of iron homeostasis in the Strategy I model plant Arabidopsis thaliana: a functional genomic approach (Chapter 2 and Chapter 3) and a mutant screen (Chapter 4). For the functional genomic approach, Affymetrix ATH-1 microarrays were hybridized with RNA extracted from shoots and roots of Arabidopsis thaliana Col-0 plants grown under iron-sufficient or -deficient conditions. The resulting datasets were screened, analyzed, and ten genes were chosen for further studies. In Chapter 2, we describe the initial characterization of eight of the ten genes, which included a metallothionein, a putative sugar transporter, three P-Type ATPases, and three vii cytochrome P450s. None of these genes appeared to have a direct effect on iron homeostasis in Arabidopsis. We also present possible future research projects using the microarray dataset as a starting point. In Chapter 3, we show the initial characterization of the other two genes selected from the microarray dataset. Both belong to a small sub-family of four genes of the basic helix-loop-helix transcription factor family of genes. All four were up-regulated in shoots and roots of iron-deficient plants. We focused our research on bHLH101, which was expressed in root and shoot meristems, in cotyledons, and in the root vasculature. Since individual T-DNA lines did not show a visible phenotype under the conditions tested, and all four show similar increases in mRNA levels under iron-deficiency, we hypothesized that all four TFs provide redundancy to each other, possibly due to homo- or hetero- dimerizing within the group. At the end of Chapter 3, we propose possible functions of these transcription factors. Finally, in Chapter 4, we show the screening of mutant plants with potential disruptions in iron-homeostasis system. The selected mutant showed variegated chlorosis and delayed induction of iron deficiency-inducible ferric-chelate reductase activity. The mutation was mapped to locus At2g34740, which