Thesis (Ph. D.)--University of Kentucky, 2004. / Includes abstract and vita. Includes bibliographical references (p. 191-198). Also available in PDF format via internet.

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Thesis (Ph. D.)--University of Kentucky, 2004. / Title from document title page (viewed Sept. 10, 2004). Document formatted into pages; contains x, 203 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 191-198).

Thesis(M.S.)--Auburn University, 2006. / Abstract. Vita. Includes bibliographic references.

Plant form is a product of emergent processes of cell division, patterning and morphogenesis. These fundamental processes remain poorly characterised in plants. However, engineering approaches can provide new tools and frameworks for the study and manipulation of plant development. This dissertation describes the development of engineering frameworks for reprogramming of the early diverging land plant Marchantia polymorpha (Marchantia). I describe the generation of genomic and transcriptomic datasets for Marchantia, which has provided the basis for the compilation of a gene-centric registry of DNA parts for engineering (MarpoDB). I describe the development of Loop assembly, an efficient and standardised DNA assembly system based on Type IIS restriction enzymes for recursive fabrication of DNA circuits with high efficiency. MarpoDB was used to mine new DNA parts compatible with Loop assembly which were used to generate plant transformation vectors for labelling of cellular features to study aspects of growth and development. I performed image analysis of genetic markers for segmentation and quantification of cellular properties in germinating gemmae. I implemented high-efficiency Cas9-mediated mutagenesis in Marchantia for use in functional molecular genetics studies. Furthermore, I produced inducible systems for expression of heterologous elements by transactivation which showed negligible levels of basal activity. It was possible to use this system for induction of gene expression in single cells. Finally, these new frameworks were applied to study the gametophytic meristem in Marchantia gemmae. I mapped the expression of several putative candidate homologues for higher plant meristem regulators, performed overexpression and loss-of-function studies for homologues of WUSCHEL, CLAVATA3 and SHOOT MERISTEMLESS. A strategy for misregulation of endogenous genes was developed using inducible transactivation, and was used with cellular markers for WUSCHEL and CLAVATA3 homologues in Marchantia.

Despite the two-fold advantage to asexual reproduction and its prevalence in a variety of organisms, sexual reproduction is prevalent across all taxa. The maintenance of two sexes is required to ensure genetic diversity and to prevent “evolutionary dead ends,” especially in clonal organisms. Many mechanisms have been proposed for the maintenance of two sexes, ranging from environmental variation and stochasticity, parasites and predators, and mutation rates. Spatial configuration, the size and location of populations with respect to other populations, can allow two competitors to coexist when one would normally be lost. This is especially important when the two competitors are the two sexes. In the clonal organism Marchantia inflexa, I determined that spatial configuration of populations can directly influence the maintenance of both sexes in a population and in an aggregate of populations (a metapopulation) using a combination of theoretical models and field studies. Based on field studies, population size has a significant influence on whether a subpopulation will contain both sexes, with populations smaller than 1m2 being more likely to contain only one sex while populations greater than 1m2 are more likely to contain both sexes. Based on mathematical models, the spatial arrangement of subpopulations within a metapopulation can greatly influence whether a metapopulation maintains both sexes as well as whether the metapopulation persists once one sex has been lost. Field data suggest that distance to nearest neighbor, a measurement of spatial arrangement, influences the maintenance of the sexes within subpopulations, but could affect maintenance differently depending on the metapopulation identity. In some metapopulations, both sexes are maintained when the nearest neighbor is close, while in other streams, one sex is lost when the nearest neighbor is close. When mathematical models are used to explicitly simulate natural metapopulations, the mathematical model predicts the observed sex ratios in one metapopulation, predicts the observed bias in another metapopulation, and fails to predicted observed values in two other metapopulations. Understanding spatial configuration helps us understand the maintenance and loss of sex, but other factors, such as environmental differences, may be required to accurately predict which sex will be lost.

The colonization of the arid continental surface by plants was one of the milestones in Earth's history. Morphological innovations, such as the origin of complex 3D tissues, allowed the successful colonization and radiation of plants on land. The epidermis is the outermost plant tissue that constitutes the interface between the plant and the environment. Thus, the evolution of epidermal cells was crucial for the adaptation of plants on the terrestrial arid environment. I undertook a combined approach that aims to understand the evolutionary trends that drove land plant colonization and the genetic mechanisms that underlie the development of the epidermis. This approach includes: 1) analyses of plant transcription factors (TFs) families distribution and diversification, with a particular focus on the basic Helix-Loop-Helix (bHLH) TF family, and 2) functional characterization of a putatively conserved bHLH TF subfamily involved in epidermal cell development in land plants. Here, I showed that there was a stepwise increase in the number of transcription factor (TF) families and bHLH subfamilies that predated the colonization of the terrestrial surface by plants. The subsequent increase in TF number on land was through duplication within pre-existing TF families and subfamilies. Moreover, a similar trend occurred in metazoan bHLH TF, suggesting that the majority of innovation in plant and metazoan TF families occurred in the Precambrian before the Phanerozoic radiation of land plants and metazoans. Furthermore, I demonstrated that the function of IIIf bHLH TFs in controlling the development of the epidermal cell layer is conserved between liverworts and angiosperms. This suggests that IIIf bHLH TFs are ancient and conserved regulators of epidermal cell development since the early colonization of the land by plants. Moreover, these bHLH TFs were recruited during the evolution of land plants to control the development of seemingly unrelated morphological characters in specific lineages of extant land plants. The recruitment of ancient developmental regulators to control distinct and unrelated developmental processes in land plants might underlie the huge morphological and taxonomic radiation of plants on land.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24982号 / 生博第511号 / 新制||生||68(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 河内 孝之, 教授 荒木 崇, 教授 中野 雄司 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

All plants have a protection against the surrounding environment called a cuticle coating. When this cuticle coating is constructed it is believed that the family of protein called lipid transfer proteins (LTPs) is involved. The LTPs are small and cysteine rich. In Marchantia polymorpha the groups of LTPs called LTPd and LTPg can be found. 8 and 4 in each group respectively. In the genes of LTPd there is an intron placed downstream of the start codon. Firstly, a sequence database search was performed and LTPd2 and LTPd3 were chosen for further experiments in this study. Secondly, a control that the intron was present in the samples were done by preforming a PCR reaction of cDNA from isolated RNA taken from untreated Marchantia polymorpha. A gel electrophoresis of the product was also performed. Lastly, the amount of alternative splicing in LTPd2 and LTPd3 from Marchantia polymorpha after treated with cold and dehydration were studied using quantitative PCR. For the qPCR MpACT and the exon of respective gene were used as references. The ΔCt values and the expression fold (2ΔΔCt) calculated from the qPCR results showed that most of the transcript with introns preserved were upregulated after subjected to stress. Only the intron in MpLTPd2 and MpLTPd3 with MpACT as reference showed a small downregulation after the cold treatment. The intron in MpLTPd3 with MpLTPd3s exon as reference didn’t show any difference. None of the intron transcript in any of the genes on the other hand showed any significant difference in the alternative splicing. This could be because of small sample groups when the test was performed. In conclusion, there were no significant difference in intron expression between treated and control samples. Therefore, nothing can be said about the change in alternative splicing in MpLTPds after cold and dehydration treatments.

Plants are threatened by global change, increasing variability in weather patterns, and associated abiotic stress. Consequently, there is an urgent need to enhance our ability to predict plant community dynamics, shifts in species distributions, and physiological responses to environmental challenges. By building a fundamental understanding of plant stress tolerance, it may be possibly to protect the ecological services, economic industries, and communities that depend on plants. Dehydration tolerance (DhT) is an important mechanism of water stress tolerance with promising translational applications. Here, I take advantage natural variation in DhT to gain a deeper insight into this complex trait. In addition, I address questions related to the causes and consequences of sexual dimorphisms in DhT. Understanding sexual dimorphisms in stress tolerance is critical because these dimorphisms can drive spatial segregation of the sexes, biased sex ratios, and may ultimately reduce sexual reproduction and population persistence. This work takes an integrated approach, addressing DhT on multiple scales from ecology, to physiology, to genomics in the tropical liverwort Marchantia inflexa. Initially, I tested for correlations between DhT and environmental dryness, sex differences in DhT, and genetic vs. plastic contributions to DhT variability. I found that patterns of variation in DhT are associated with environmental variability, including complex sexual dimorphisms, and derive from a combination of plasticity and genetic differences in DhT. Subsequently, I leveraged the variability in DhT to identify candidate DhT enhancing genes. In M. inflexa intraspecific differences in DhT are impacted by baseline variability among plants, as well as unique gene expression responses initiated during drying. In parallel, I assembled a draft genome assembly for M. inflexa, which was employed to investigate questions of sex chromosome evolution and sexual dimorphism in DhT. Finally, the bacteriome of M. inflexa was characterized and found to be extremely diverse and variable. Collectively, this work adds to a growing understanding of DhT and highlights the importance of sampling approaches that seek to comprehensively describe variability in DhT. I detected complex patterns of variability in DhT among populations and the sexes of M. inflexa, which were used to gain insight into the genetic intricacies of DhT.