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The Pennsylvania State University. The Graduate School. Department of Biology INVESTIGATING EVOLUTION OF PLANT DEVELOPMENT IN BASAL ANGIOSPERMS



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The Pennsylvania State University The Graduate School Department of Biology INVESTIGATING EVOLUTION OF PLANT DEVELOPMENT IN BASAL ANGIOSPERMS A Dissertation in Biology by Barbara Joanne Bliss 2008 Barbara
The Pennsylvania State University The Graduate School Department of Biology INVESTIGATING EVOLUTION OF PLANT DEVELOPMENT IN BASAL ANGIOSPERMS A Dissertation in Biology by Barbara Joanne Bliss 2008 Barbara Joanne Bliss Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2008 The dissertation of Barbara Bliss was reviewed and approved* by the following: Hong Ma Professor of Biology Dissertation Co-Advisor Co-chair of Committee Claude depamphilis Professor of Biology Dissertation Co-Advisor Co-chair of Committee Paula McSteen Assistant Professor Siela Maximova Research Associate Doug Cavener Professor of Biology Head of the Department of Biology *Signatures are on file in the Graduate School iii ABSTRACT Understanding the evolution of modern plants requires integrating findings from several disciplines, including plant physiology and development, molecular genetics, and genomics. Observations from model and non-model plants are brought together in a phylogenetic framework to derive hypotheses about how plant development evolved to generate the abundant diversity we see today. Testing those hypotheses requires a plant model system with the appropriate phylogenetic perspective: that of a basal lineage. The greatest diversity of plants today is among the angiosperms (flowering plants), a lineage which arose only about 160 million years ago. The most successful of these are the monocot and core eudicot flowering plant lineages, from which current plant model experimental systems are derived. For questions about the evolution of angiosperm development, a plant model from among the basal lineages is required. In addition to phylogenetic perspective, model systems possess features and degrees of availability, representation, and utility not found in other members of the taxa to which they belong. For all organisms, culturing requirements are central determinants of utility, but for studying the evolution of plant development, amenability to studies employing methods of genomics, genetics, molecular and developmental biology are also required. This dissertation describes the search for and selection of a proposed basal angiosperm experimental model, Aristolochia fimbriata, along with the development of initial technologies required for testing hypotheses about the evolution of plant iv development. Culturing, hand pollination, genetic transformation, and in vitro micropropagation and regeneration methods are described herein. Genes involved in flower form and architecture have been particularly important in the evolution of angiosperm diversity. The TCP gene family, so named for its founding members (TEOSINTE BRANCHED1, CYCLOIDEA, PROLIFERATING CELL FACTOR) has been shown to play important roles in evolution of form in both monocots and eudicots. Prior functional and phylogenetic analyses of this gene family revealed clades of TCP genes with two different kinds of gene function. Since then, additional sequence data from basal lineages and new studies providing insight into TCP gene function have become available. Together, these warrant an updated phylogeny and review of this important gene family. Preliminary phylogenetic analyses of the TCP gene family is described as a foundation for conducting future expression and functional analyses. A. fimbriata has floral and vegetative features that will facilitate evaluating the role of TCP genes in evolution of angiosperm form, and advance the use of this species as a basal angiosperm model system. v TABLE OF CONTENTS LIST OF FIGURES... ix LIST OF TABLES... xii LIST OF ABBREVIATIONS... xiv ACKNOWLEDGEMENTS... xvii Chapter 1 Introduction... 1 Innovations in land plants... 2 Bryophytes and tracheophytes: Controlling water and gravity... 2 Spermatophytes: Efficient reproduction... 6 Angiosperms: Flowering plants... 8 Angiosperm phylogeny Phylogenetic analysis Selection of sequence data Basal angiosperms Tracheary elements Double fertilization Flower Floral developmental genetics Floral organ identity model Genes involved in floral organ identity Broader applications Basal angiosperm model Transformation systems Regeneration systems Overview of dissertation Chapter 2 Aristolochia fimbriata: A proposed experimental model for basal angiosperms Abstract Introduction Results Evaluation of potential models in basal angiosperm orders and families Aristolochiaceae candidates considered Physical and life cycle features of Aristolochia fimbriata A phylogenetic perspective of genome sizes Methods for genetics Discussion... 60 vi Aristolochia fimbriata has many characteristics of a valuable experimental model Aristolochia fimbriata is well positioned for studies of the evolution of development Aristolochia contains highly developed biochemical pathways offering insight into evolution of biochemical synthesis and coevolution with insects Aristolochia can provide insight into development of woodiness Aristolochia can reveal features of the ancestor common to monocots and eudicots Growing genomic resources in Aristolochiaceae support further development of model system Conclusion Materials & Methods Cultivation Genome sizing Phylogenetic analysis Pollination experiments Genetic transformation Genomic PCR analysis Genetic transformation Supplemental Material Acknowledgements Cultivation Supplement Background Perianth maturation Hand pollination methods Pollination is prevented with pollination bags Day two autogamous pollinations are most successful Self-compatibility in Aristolochia elegans Seed germination methods Effect of seed age on germination Chapter 3 In vitro propagation, shoot regeneration and rooting protocols for Aristolochia fimbriata, potential model plant for basal angiosperms Abstract Introduction Materials and Methods Plant material Micropropagation initiation and multiplication In vitro rooting Shoot regeneration from whole leaf explants Shoot regeneration from petiole and stem sections Results Micropropagation and in vitro rooting Shoot regeneration Discussion Conclusions Acknowledgements Chapter 4 TCP gene family evolution Founding members: TB1, CYC, and PCF TCP gene functions Class I TCP proteins Class II TCP proteins Class III TCP proteins Evolution by changing gene interactions Regulation by methylation Materials and Methods Automated pipeline Verification of sequences Results Sequence collection Alignments Phylogenetic analysis Discussion Chapter 5 Retrospective and future direction for studies of evolution of development in basal angiosperms Improving the Aristolochia fimbriata transformation system Evolution of gene function in basal angiosperms Appendix A Whole genome comparisons reveal intergenomic transfers Abstract Introduction Methods Sequence selection, alignment, and phylogenetic analysis Comparing distributions of frequencies of similarity scores Amplification and sequencing Results Discussion Acknowlegements Appendix B Cultivation Supplement Seed germination experiments Germinating in toweling vii Seed storage experiments Inducing flowering Hand pollination Recording pedigree Example 1: Example 2: Possible mutants Literature cited viii ix LIST OF FIGURES Figure 1-1: Evolution of major innovations in embryophytes Figure 1-2: Angiosperm flowers Figure 1-3: A consensus phylogeny of angiosperms Figure 1-4: Floral specializations Figure 2-1: Angiosperm phylogeny based on Stevens (2007) (http://www.mobot.org/mobot/research/apweb/welcome.html) Figure 2-2: Overall approach for selecting a basal angiosperm model system Figure 2-3: Genome sizes in basal angiosperm families updated with Cui et al. (2006) and Bennett and Leitch (2005), shown on logarithmic scale Figure 2-4: Diversity of flower and growth forms in Aristolochiaceae Figure 2-5: Aristolochia fimbriata Figure 2-6: Phylogenetic relationships among sampled Aristolochiaceae, with Tasmannia lanceolata (Cannelleales) as outgroup Figure 2-7: Green fluorescent protein expression in Aristolochiaceae Figure 2S-1: Aristolochia fimbriata flower Figure 2S-2: Hand pollination, A. fimbriata Figure 2S-3: Aristolochia fimbriata genotype and perianth detail Figure 2S-4: Phylogram of Aristolochiaceae relationships, showing minimal variation in branch lengths within Aristolochiaceae Figure 3-1: Aristolochia fimbriata regeneration Figure 3-2: Schematic representation of micropropagation and regeneration protocols of A. fimbriata Figure 4-1: Supertribe 777, MUSCLE alignment of amino acids Figure 4-2: Supertribe 1148, MUSCLE alignment of amino acids x Figure 4-3: Supertribe 2066, MUSCLE alignment of amino acids Figure 4-4: RaxML phylogenetic analysis of TCP domain-containing sequences from automated pipeline Figure 4-5: Neighbor joining tree and expression pattern of TCP genes in rice and Arabidopsis from Yao et al. (2007) Figure 4-6: Unrooted ML tree of 126 TCP proteins from land plants from (Navaud et al., 2007) Figure 4-7: Classes 1, 2, 3 TCP sequences Figure 4-8: Class 1 (PCF-like) TCP sequences Figure 4-9: HMM logo PFAM Figure 5-1: Evaluating Kanamycin (Kan) as selection agent Figure 5-2: GFP expression in A. fimbriata roots Figure A-1: MultiPipMaker plots of organelle genome sequences aligned to chloroplast target genome sequences Figure A-2: Frequency of high identity regions in alignments of real and simulated mitochondrial to chloroplast genome sequences Figure A-3: Distribution of similarity scores for 100 bp windows from 17 pairwise alignments of mt genome sequence or simulated mt genome sequence to cp genome sequence Figure A-4: Strict consensus trees from NJ analysis with PAUP* (500 bootstrap replicates) using aligned coding sequences from cp and mt genomes Figure A-5: Successful amplification of psba (A, C), rbcl (B, D), and ycf1 (E, F), from the Arabidopsis thaliana (A, B, E) chloroplast and (C, D, F) mitochondrial genomes Figure A-6: Strict consensus trees from PAUP* (500 bootstrap replicates) analysis of aligned coding sequences from cp and mt genomes Figure B-1: Seeds planted in varying conditions on 9/28/ Figure B-2: Comparing effects of light on germination of seeds planted on ½ MS media Figure B-3: Comparing seed germination on wet filter paper or medium Figure B-4: Comparing effect of light on germination of seeds planted in wet toweling Figure B-5: Effects of light on germination of seeds planted in wet toweling Figure B-6: Comparing effect of light on germination of seeds planted on soft media Figure B-7: Toweling germination method Figure B-8: Short term effects of seed age on germination Figure B-9: Longer-term effects of seed age on germination Figure B-10: Inducing blooms Figure B-11: A. fimbriata produces successive axillary blooms along the shoot Figure B-12: A. fimbriata in bloom Figure B-13: Pollination bag Figure B-14: Pollination bag attachment detail Figure B-15: Day of anthesis Figure B-16: Gynostemium Figure B-17: Self-pollination in a day two flower Figure B-18: Inferior ovary Figure B-19: Abnormal seedlings xi xii LIST OF TABLES Table 2-1: Homologs of genes involved in development, cell wall biosynthesis, and response to biotic and abiotic stress revealed in preliminary sequencing of two cdna libraries constructed from combined A. fimbriata tissues Table 2S-1: A summary of relevant basal angiosperm characteristics Table 2S-2: Cultivation features of 25 Aristolochiaceae taxa evaluated, with findings Table 2S-3: Evaluating self-compatibility in A. fimbriata Table 2S-4: Percent fruit set (sample size) in A. elegans resulting from hand pollinations of flowers on days 1-4 with autogamous, geitonogamous and xenogamous pollen from flowers on days Table 2S-5: Genome sizes, vouchers, sources and accessions for sequence data used Table 3-1: Media formulations for micropropagation and shoot organogenesis Table 3-2: The effect of plant growth regulators on axillary shoot proliferation at 21 days after culture initiation Table 3-3: Effect of indole-3-butyric acid (IBA) on rooting of shoots multiplied in vitro Table 3-4: Direct organogenesis of Aristolochia fimbriata from whole leaf explants Table 3-5: Shoot regeneration of petiole and stem explants Table 3-6: Shoot regeneration, rooting and acclimation of plants regenerated from petiole and stem explants from plants maintained in REN2 medium Table 4-1: Arabidopsis thaliana (At) and Oryza sativa ssp. japonica (Os) TCP genes from Yao et al. (2007) and Navaud et al. (2007) Table 4-2: Three supertribes of genomic sequences comprise Arabidopsis and rice genes of interest Table 4-3: Genomic sequences containing TCP domain (PFAM domain 03634) Table A-1: List of species and GenBank accessions for 25 organelle genome sequences Table A-2: Primers to amplify chloroplast genes from Arabidopsis cp and mt genomes Table A-3: Locations of chloroplast-specific sequences in pairwise alignments of mt genome sequences with cp genome sequences Table B-1: Pedigree record, example Table B-2: Pedigree record, example Table B-3: Pedigree and incidence of tricots xiii xiv LIST OF ABBREVIATIONS 6BA: 6-benzoamino-purine AAGP: Ancestral Angiosperm Genome Project (NSF DEB ) AG: AGAMOUS AGL6: AGAMOUS-Like 6 AP1: APETAL1 bp: base pairs CaMV: cauliflower mosaic virus cp: chloroplast CUC: CUP-SHAPED COTYLEDON cup: cupuliformis CYC: CYCLOIDEA DICH: DICHOTOMA DIV: DIVARICATA EGFP: enhanced green fluorescent protein ERF: ethylene responsive factor EST: expressed sequence tags FGP: Floral Genome Project (NSF DBI ) GFP: green fluorescent protein HGT: Horizontal gene transfer IBA: indole-3-butyric acid Kan: kanamycin MADS: MCM, AGAMOUS, DEFICIENS, SRFC MAX: MORE AXILLARY GROWTH xv MI: micropropagation initiation medium MP: micropropagation medium MS: Murashige and Skoog (basal medium) mt: mitochondrial mya: million years ago NAA: -naphthalene acetic acid nptii: neomycin phosphotransferase orf: open reading frame PCF: proliferating cell factor PCNA: proliferating cell nuclear antigen PCR: polymerase chain reaction petg: cytochrome b6/f complex subunit 5 PGR: plant growth regulator pip: percent identity plot PGR: plant growth regulator psaa: photosystem I P700 apoprotein A1 psba: photosystem II reaction center polypeptide D1 psbd: photosystem II reaction center protein D2 QTL: quantitative trait loci rbcl: ribulose 1, 5 bisphosphate carboxylase/oxygenease, large subunit RAD: RADIALIS REN1, REN2: root elongation media one and two RI: root initiation medium SI: shoot induction medium xvi SR: shoot regeneration medium TB1: TEOSINTE BRANCHED1 TCP: TB1, CYC, PCF TDZ: thidiazuron ycf1, ycf2: large orf common in angiosperm chloroplast genomes xvii ACKNOWLEDGEMENTS I would like to thank my committee, for their individual support and collective service on my behalf. In the lab of Dr. Claude depamphilis, I conceived of my project and brought it to bear, a feat I certainly could have done nowhere else. The hand of Dr. Hong Ma kept me guided firmly toward my goal; providing me with technical expertise and supporting my interest in classical and molecular genetics. Dr. Siela Maximova has been an unfailing inspiration, even when all else failed, and Dr. Paula McSteen has served to moderate and tell me plainly what the outcome should look like. My work at Penn State was supported in by a University Graduate Fellowship from the graduate school of Penn State University and a Braddock Fellowship from the Eberly College of Science. During , the work was supported by NSF grants DBI (Floral Genome Project) and DEB (Ancestral Angiosperm Genome Project) to Claude depamphilis, Teaching Assistantships from the Department of Biology, and travel grants from the Floral Genome Project. I received another Braddock Fellowship from the Eberly College of Science in I deeply appreciate my colleagues and mentors. My lab mates Jill R. Duarte, Yan Zhang, and most especially Joel McNeal shared their expertise. Jim Leebens-Mack, Kerr Wall, Paula Ralph, and Ali Barakat provided the eagle s perspective, and Laura Zahn contributed the outsider s viewpoint, encouragement, and friendship. My training has also been shaped by the excellent habits of Sharon Pishak and the focus and determination of Stefan Wanke. xviii I am grateful to my family and friends who recklessly urged me to embark on this endeavor, knowing I would not rest until I had done so. They have not ceased to remind me that despite circumstances encountered along the way, this was - and is - the right path for me. I especially want to acknowledge the sacrifices of my two children, who gave up so much of Mom just so that she could go to school to get a job that she liked. I hope they, too, will not hesitate to go to school to get a job that they like, and that if they are also parents when they do, that their children give them just as hard a time as they have given me. They have been the wind beneath my wings; which has been, indeed, the wind of a firestorm threatening me with certain immolation if I do not rise high, quickly enough Chapter 1 Introduction Research into the evolution of plant development aims to understand the mechanisms which have given rise to the wide variety of plants we observe today (Raff, 2000). The most speciose extant plant lineage is that of the angiosperms, with over 250,000 species. In the angiosperms, the innovations of flower, fruit, and double fertilization supported such a rapid radiation of diversity that Darwin described it as an abominable mystery (1903). Although the origins of the angiosperms have seemed enigmatic, as the disciplines of developmental biology (from early embryology), genetics, molecular biology, molecular evolution, and modern genomics are brought to bear, Darwin s abominable mystery is giving way to a rudimentary understanding of processes involved in the evolution of angiosperm development (Friedman et al., 2004). 2 Innovations in land plants Ontogeny (embryology or the development of the individual) is a concise and compressed recapitulation of phylogeny (the paleontological or genealogical series) conditioned by laws of heredity and adaptation. (Haeckel, 1909) The evolution of development that led to the flowering plant lineage was directed and limited by development that had occurred previously, and had proved to be advantageous. Development in an individual flowering plant does not strictly recapitulate the evolutionary history of angiosperms, but major changes in form and organization can be identified in the lineage leading to land plants (embryophytes) since they last shared a common ancestor with charophycean algae (Endress, 2006; Qiu et al., 2007). Thus, it is necessary to study the predecessors of flowering plants in order to recognize and construct hypotheses about what features evolved to give rise to the flowering plants. What follows is a brief review of the major vegetative and reproductive innovations in land plants leading to the success of the angiosperm lineage. Bryophytes and tracheophytes: Controlling water and gravity Early adaptive changes required for successful colonization of land arose around 480 million years ago (mya) in the Ordovician period, in an aquatic common ancestor the embryophytes shared with Characeae (Karol et al., 2001; Judd et al., 2002; Simpson, 2006). These early adaptations included apical development and phragmoplastic cell division, necessary for producing land plant architecture. Cellulosic cell walls and specialized gametangia (antheridia and archegonia) with encased egg cells (cortication) 3 contributed to the ability to survive drying conditions of land, particularly for reproductive structures (Figure 1-1). Land plants appeared in the lower Silurian (approx 430 mya) and shared further adaptations, including a cuticle and jacketed gametangia to further protect the gametes and embryo from desiccation (Figure 1-1) (Qiu, 2008). The basal nonvascular embryophytes are known as the bryophytes and include the mosses, hornworts, and liverworts (Figure 1-1) (Nickrent et al., 2000). The genome sequence of the moss Physcomitrella patens
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