Seed dormancy is a mechanism with great importance in plant fitness and it inhibits seed germination until is broken and seeds can germinate under optimal environmental conditions favorable for successful reproduction. Primary dormancy is contingent to the environment that seeds and the mother plant experience. Temperature is a major factor participating in the regulation of this complex trait. High and low levels of dormancy are induced during seed maturation by cold and warm temperatures respectively but the mechanism at the basis of temperature signaling in seeds is not well understood. Climate change and increased weather variability threaten the constant supply of high quality seeds into the market hence agriculture productivity. Therefore, understanding and taking control of the molecular mechanism behind the regulation of seed dormancy and germination will help to control and predict seed behavior in the field. Here I describe and discuss a forward genetic screen for the selection of mutant seed lines with altered seed dormancy in response to cool temperature during seed set. Putative mutant seed lines designated awake1 to awake52, were preliminarily characterized. Eleven awake lines were selected for further analysis and one was investigated in more detail. It was revealed that awake1 seeds shares common phenotype with seeds of a suberin deficient mutant which were previously reported to display increased dormancy but, here, I show they also display a reduction of seed dormancy. Segregation analysis suggests that the reduced dormancy phenotype is maternally inherited as the suberin deficient mutants. Also, transcriptomic analysis shows that many suberin associated genes are temperature-regulated. I conclude that control of suberin deposition may play a role in the regulation of dormancy in response to cool temperature.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19069号 / 人博第722号 / 新制||人||173 / 32020 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 宮下 英明, 教授 川本 卓男 / 学位規則第4条第1項該当

Plant growth and development depends on controlled cell expansion. This, in itself, is determined by the plant cell wall, a structural matrix of polysaccharides encasing the plant cell. One line of investigation that has proven particularly successful in elucidating the components of the plant cell wall machinery has been the forward genetic screens of cell wall mutants. In this study, the molecular and cellular characterisation of sloughy, a cell separation mutant in Arabidopsis thaliana, was commenced. This mutant has a striking phenotype, with files of elongating epidermal cells snaking away from the adjacent epidermal cells and from the underlying cortex, loosing contact from the side walls while remaining attached at the cell ends, in a manner reminiscent of border-like cells in the root cap of arabidopsis. The sloughy mutation was fine mapped to a short region on chromosome I using high resolution melt point analysis. On sequencing all five genes in this region, a single nucleotide mutation, introducing a stop codon, was detected in exon 2 in the previously-described heat shock transcription factor SCHIZORIZA that results in a truncated protein missing several conserved domains essential for activity. SCHIZORIZA acts as a cell fate determinate in the root meristem to promote cortex fate, while suppressing epidermal and root cap fate in the mature ground tissue. Although the literature on schizoriza mutants has focused on the developing root meristem, with little documentation on the cell separation phenotype further up in the roots, the investigation of a collection of schizoriza TILLING mutants revealed that aberrant cell separation was ubiquitous to schizoriza mutants with a severely truncated protein. To investigate cell identity in the mature roots, sloughy was crossed to GAL4-GFP enhancer trap lines that act as cell-specific markers. Epidermal identity lines revealed that sloughy possessed a supernumerary ground tissue layer with epidermal identity. A cortex and endodermal line revealed that these two identities are restricted to the endodermal layer and the next ground tissue layer out. There was no indication of root cap identity in the mature root with any of the root cap lines used, although partial lateral root cap identity has been previously described in the epidermal and subepidermal cell layers in the meristem of schizoriza mutants expressing SOMBRERO-GFP, a lateral root cap-specific transcription factor. Immunolabelling of cell wall epitopes revealed that the JIM13 antibody, which specifically labels arabinogalactan-proteins in wild-type root caps, often labelled the epidermal cells and surrounding mucilage further up the in the roots of sloughy. The aberrant cell separation present in sloughy is thought to be a consequence of epidermal cells possessing partial lateral root cap identity. The data on sloughy/schizoriza is sufficient to generate a model on how a meristem developmental gene can generate a cell separation phenotype in the mature roots. Loss of SCHIZORIZA causes confused cell identity in the root meristem that results in an epidermal and subepidermal layer possessing mixed epidermal and lateral root cap identity. The distinctive properties of border-like cells in the root cap of arabidopsis have been linked to unique cell wall maturation and developmental processes, implicating the cellulases CEL3 and CEL5, the pectin glycosyltransferase QUA1, the pectin methyltransferase QUA2 and other pectolytic enzymes. The ectopic expression of these cell wall enzymes in the epidermal and subepidermal layers of sloughy roots result in reduced adhesion along the sides of the cell, while the ends remain attached, causing the observed cell separation phenotype.

Many of the cutting-edge innovations on the CRISPR/Cas9 system involve the use of complex sgRNA libraries. For example, such libraries have recently been used in functional genomics applications to screen for specific genes in both cell lines and living organisms, as well as in subcellular imaging to fluorescently label chromatin in living cells. However, chemically synthesizing customized libraries can be cost prohibitive, requires precise genomic information, and can takes several weeks. We developed a rapid and efficient enzymatic method to generate sgRNA libraries from arbitrary DNA substrates, significantly reducing the cost of library generation. A type IIS restriction enzyme binding site was incorporated directly into the sgRNA scaffold sequence without affecting the catalytic properties of the Cas9 complex, resulting in a simple method that generates high fidelity sgRNA libraries. Additionally, the library is constructed on the surface of streptavidin coated magnetic beads and a strand displacing polymerase is used to elute DNA from the beads, reducing the loss of material and simplifying purification between steps. We used this method to generate two sgRNA libraries. The first targets the entire E. coli genome, and the second targets genes expressed in the developing zebrafish heart. This second library will be used in a forward genetic screen in zebrafish to identify genes underlying heart defects. These complex sgRNA libraries show the utility of the method in generating libraries from DNA from any species, ranging from prokaryotes like bacteria, to eukaryotes including plants and animals.

京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19069号 / 人博第722号 / 新制||人||173(附属図書館) / 26||人博||722(吉田南総合図書館) / 32020 / 京都大学大学院人間・環境学研究科相関環境学専攻 / (主査)准教授 土屋 徹, 教授 宮下 英明, 教授 川本 卓男 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DFAM

Mutations in the telomere length maintenance pathway can lead to a spectrum of diseases called telomere syndromes, however, the pathway is not fully understood and there may still be unknown components. We designed a forward genetic screen to identify new genes involved in telomere length maintenance. Of the top ranked genes, ZNF827, a zinc finger protein, is the most promising candidate gene. The possible discovery of a new component involved in telomere length maintenance increases our understanding of the pathway and opens new avenues of research. Recent advances in molecular biology techniques, such as the use of RNA-guided nuclease CRISPR associated protein 9 (Cas9), have made screens like this possible. Cas9 is a nuclease that uses a guide RNA(gRNA) to direct its endonuclease activity. The use of Cas9 has revolutionized the field of genome engineering, providing scientists with more efficient methods to knockout and modify genomes. We sought to optimize CRISPR-Cas9 genome editing to make it as widely accessible as possible. We compared plasmid, ribonucleoprotein (RNP), and RNA only lipid-mediated transfection of CRISPR-Cas9 into cell lines using a novel reporter system to measure genome editing efficiency. All methods were successful to some extent, however, RNP lipofection was the most efficient and has many advantages over other methods. We also found that short homology arms of 30-35bp on donor templates was able to mediate site specific editing. These methods should broaden the accessibility of CRISPR-Cas9 genome editing.

Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.

Hematopoiesis is an intricately regulated homeostatic process that maintains all of the differentiated blood cell lineages. N-ethyl-N-nitrosurea (ENU) is a powerful mutagen that induces point mutations randomly in the genome. ENU was used in a dominant forward genetic screen to identify novel mutations in regulators of hematopoiesis and to create new mouse models of hematopoietic disease. The objectives of this thesis were to characterize two mutants that originated from the dominant screen (7192 and 7238) and to develop a pharmacologically sensitized screen that would detect a unique set of mutations undetectable in the dominant screen. The 7192 mutant from the ENU dominant screen presented with elevated microcytic red blood cells (RBC) and increased polychromasia. The causative mutation was identified as a nonsense mutation in Ank1 (Q895X) that coded for a truncated ANK1 protein. Ank17192 is a novel mouse model of hereditary spherocytosis (HS), a human disease that results from increased RBC fragility. We have demonstrated that Ank17192/+ mice model a mild HS and Ank17192/7192 mice model severe HS. The 7238 mutant from the dominant ENU screen was macrothrombocytic and carried a missense mutation in Myh9 (Q1443L). The Myh97238/7238 mice are viable and have a more severe phenotype of macrothrombocytopenia. Myh97238 is the first mouse model for Myh9 related disorders that accurately models the genetic origins and the systemic manifestations of the disorder. A pharmacologically sensitized screen using chemotherapeutic drugs was designed to induce stress hematopoiesis to detect mutations that alter cell cycle of hematopoietic progenitors or stress hematopoiesis. Analysis of both peripheral blood and progenitor recovery kinetics, determined that 5-fluorouracil (5FU) and phenylhydrazine were good candidates for a pharmacologically sensitized screen. 5FU was successfully incorporated into an ENU dominant screen, and 13 platelet recovery outliers were detected. From these outliers, three mutant lines were successfully established.