<p>Plants are used as a primary food source by humans. Some plants produce edible roots or leaves, but most crops used today are grown to harvest their nutrient-rich seeds which are a product of double fertilization in flowering plants. </p> <p>Cell-cell recognition, adhesion, and fusion are widespread phenomena in many biological processes, where fertilization is an exemplary process. Many players have been identified to mediate sperm-egg fusion in both animals and plants. Interestingly several of these components were shown to be structurally and functionally conserved across kingdoms. In animals Tetraspanins act as facilitators of sperm-egg fusion. Tetraspanins are known to associate in clusters in the plasma membrane of cells, where they recruit diverse signaling proteins, forming the so called Tetraspanin-enriched microdomains (TEMs). TEMs are therefore recognized as major signaling platforms mediating specific cellular processes in the plasma membrane of cells. Two <i>Arabidopsis</i>-expressed tetraspanins, <i>TET11</i> and <i>TET12</i>, are highly expressed in the sperm cells (SCs), however their function in fertilization are unknown. Using fluorescence microscopy, we quantified the expression of TETs in SCs and found evidence for the existence of a Tetraspanin-enriched microdomain (TEM) at the SC-SC adhesion interface. Sperm cell factors which are necessary for fertilization were found to accumulate at the TEM, suggesting that plant SC TEMs may function as protective platforms for fertilization factors. Sperm-expressed TETs directly interact with members of a novel, plant-specific family of unknown proteins, <i>DMP8/9</i>. DMP8/9 function as negative regulators of SC-SC adhesion and are required for double fertilization. Structural and functional analysis suggest that these two proteins may perform unique functions as membrane remodelers in SCs. In addition, we provide evidence of a new GEX2 function as a SC-SC adhesion factor and potential partner of TET-DMP complexes at the SC-SC interface.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18421号 / 生博第301号 / 新制||生||40(附属図書館) / 31279 / 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 佐藤 文彦, 教授 永尾 雅哉, 教授 福澤 秀哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

No description available.

The regulation of pH is a critical homeostatic function of plant cells. In addition to acting as the primary cationic species responsible for energizing the plasma membrane, protons likely act as an important regulator and messenger. Despite this importance, few studies have thoroughly described cytosolic pH patterns as the plant cell progresses through the cell cycle. To investigate pH in plant cells, I chose Nicotiana tabacum (tobacco) Bright Yellow-2 (BY-2) cells as a model system. My research has two aims. First, I will measure and report the interphase cytosolic pH of BY-2 cells. Next, I will assay the cytosolic pH as BY-2 cells progress through mitosis and cytokinesis. I hypothesize that pH patterns are be temporally or spatially associated with structures such as the mitotic spindle or the phragmoplast. To investigate cytosolic pH in BY-2 cells, I will develop a cytosolic pH reporter based on a pH sensitive ratiometric fluorescent dye. This dye will be able to resolve both temporal and spatial changes in pH throughout the cytosol while imposing a minimal amount of stress on BY-2 cells. I found that pH-GFP, a variant of eGFP, had qualities of a robust pH reporter. To introduce the dye, explored biolistic bombardment, Agrobacterium mediated transient transformation, and polyethylene glycol mediated transformation as methods for introducing the pH-GFP gene into BY-2 cells. I observed very few transformation events using these methods and my observations did not support these approaches as suitable for introducing pH-GFP into BY-2 cells.

Plant cell culture is an attractive platform technology for production and supply of important plant derived medicinals. A unique characteristic of plant cells is the ability to grow as multicellular aggregates in suspension. The presence of these non-uniform aggregates results in creation of distinct microenvironments, which can induce variations in cellular metabolism (e.g., growth, oxygen consumption and secondary metabolite synthesis). This heterogeneity can lead to unpredictable and suboptimal performance in large scale bioprocesses. One example is the Taxus cell culture system, which produces a widely used chemotherapeutic drug - paclitaxel (Taxol ®). Despite extensive process engineering efforts which have led to increased yields of paclitaxel, Taxus cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces the accumulation of paclitaxel, but to varying extents in culture. A significant negative correlation was observed between paclitaxel level and mean aggregate size of the culture, demonstrating the relevance of measuring, and potentially controlling aggregate size during long term subculture. Understanding the regulation of gene expression can provide rational engineering strategies to control variability and optimize performance of Taxus cell cultures. Biosynthetic pathway gene analyses revealed upregulation of genes upon elicitation with MeJA; results also suggested additional molecular regulatory points outside of the biosynthetic pathway. In order to fully understand Taxus molecular regulation and the relationship to paclitaxel production variability, a transcriptome-wide analysis using next generation sequencing (454 and Illumina) methods was performed. Several pathways outside of paclitaxel biosynthesis were found active upon MeJA elicitation. Global comparison of gene expression amongst cultures accumulating different levels of paclitaxel is being performed to completely understand the interactions amongst the paclitaxel biosynthetic pathway and other complimentary and competing pathways to suggest effective targets for metabolic engineering. This work collectively represents the first molecular studies to understand metabolic regulation in Taxus cell cultures. Apart from inducing paclitaxel biosynthesis, MeJA decreases cell growth in Taxus cell cultures. The MeJA-mediated repression of cell growth was shown to correlate with inhibition of cell cycle progression as evident both at the culture level through flow cytometric analyses and at the transcriptional level by repression of key cell cycle-associated genes. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.

Plant derived natural products represent some of the most effective anti-cancer and anti-infectious disease pharmaceuticals available today. However, uncertainty regarding the feasibility of commercial supply due to the limited availability of many plants in nature has resulted in a dramatic reduction in the use of natural products as leads in modern drug discovery. Plant cell suspension culture, consisting of dedifferentiated plant cells grown in vitro and amenable to large scale industrial biotechnology processes, is a production alternative which promises renewable and economical supply of these important drugs. The widespread application of this technology is limited by low product yields, slow growth rates, challenges in scale-up, and above all, variability in these properties, which is poorly understood. Plant cells grow as aggregates in suspension cultures ranging from two to thousands of cells (less than 100 micron to well over 2 mm). Aggregates have long been identified as an important feature of plant cell culture systems, as they create microenvironments for individual cells with respect to nutrient limitations, cell-cell signaling, and applied shear in the in vitro environment. Despite its purported significance, a rigorous engineering analysis of aggregation has remained elusive. In this thesis, aggregation was characterized, analyzed, and modeled in Taxus suspension cultures, which produce the anti-cancer drug paclitaxel. A technique was developed to reliably and routinely measure aggregate size using a Coulter counter. The analysis of aggregate size as a process variable was then used to evaluate the effect of aggregation on process performance, and the analysis of single cells isolated from different sized aggregates was used to understand the effect of aggregation on cellular metabolism and heterogeneity. Process characterization studies indicated that aggregate size changed over a batch cycle as well as from batch to batch, so a population balance equation model was developed to describe and predict these changes in the aggregate size distribution. This multi-scale engineering approach towards understanding plant cell aggregation serves as an important step in the development of rational strategies aimed at controlling the process variability which has heretofore limited the application of plant cell culture technology.

<p> </p> <p>Phosphorus (P) is an essential macronutrient for plant growth and development. P deficiency is becoming one of the most limiting factors for crop productivity. It has been discovered that vascular tissue-mediated systemic signaling plays important roles in plant responses to P deficient growth conditions. In order to understand vascular tissue-specific molecular alterations in response to P deficiency, I used <em>Plantago major </em>as a model species to study the transcriptomic alterations in vascular tissues because it is fast and easy to dissect pure vascular tissues from this plant. I identified 237 differentially expressed genes involved in various roles to P deficiency, such as “phosphate metabolism and remobilization”, “sucrose metabolism, loading and synthesis” and “plant hormone metabolism and signal transduction”. In addition, translating ribosome affinity purification (TRAP) was used to identify 547 differentially expressed genes from the Arabidopsis vascular tissues. <em>AtERF</em>, one of the downregulated genes, was chosen for further functional characterization. My results demonstrated that <em>AtERF </em>is specifically expressed in vascular tissues and it encodes a transcription factor. Over-expression of <em>AtERF </em>led to a purple vein phenotype, decreased growth of shoots and roots, and reduced Pi concentrations in shoots and roots. The <em>erf </em>mutant plants displayed larger shoots and roots, and increased Pi concentration in shoots and roots. Molecular analysis in the over-expression and mutant plants showed that genes related to hormone metabolisms and root architecture establishment might be the major players enabling plants to cope with low P. The discoveries from this study may be used to implement strategies for the production of crops with increased P uptake efficiency. </p>

<p dir="ltr">Plants are capable of perceiving and responding to biotic and abiotic stress. They have evolved a variety of mechanisms to help them recognize and trigger rapid responses to both chemical and mechanical stimuli. These signals coordinate plant growth, development, and innate immune responses. However, we have limited knowledge about how mechanical signals are perceived and transduced during the plant immune response. In this study, we investigated the potential role of PIEZO, a mechanosensitive ion channel that is responsible for cellular mechanotransduction in both the plant and animal kingdoms, during the plant immune responses. Publicly- available RNAseq data revealed that <i>PIEZO</i> expression remained constant and unaltered in response to a variety of phytopathogens or elicitors. We, then, conducted infectious growth assays on <i>piezo</i> mutants in <i>Arabidopsis thaliana</i> plants. Our results indicated that <i>piezo</i> mutants, <i>pzo1-1 </i>and<i> pzo1-5,</i> were more susceptible to <i>Pseudomonas syringae </i>pv. tomato<i> </i>(Pst) DC3000 and to the <i>P. syringae hrcC</i>^<em>-</em> mutant confirming PIEZO’s role in plant defense and PTI. We further explored disease progression with necrotrophic fungi, <i>Alternaria brassisicola</i> and <i>Botrytis cinerea, </i>on <i>piezo</i> mutant plants and found enhanced fungal growth compared to the wild type (Col-0) with <i>Botrytis</i>. Building upon these findings, we probed the role of PIEZO in the growth-defense tradeoff using a root growth inhibition assay with flg22 as the MAMP elicitor. <i>pzo1-1</i> was less sensitive to flg22 treatment with less reduction in root growth compared to wild type whereas <i>pzo1-5</i> shows no difference in reduction compared to Col-0. In addition, we investigated whether PIEZO operates upstream of the main NADPH-oxidase, RBOHD, and the associated oxidative burst that occurs in early defense. There was no significant difference in Reactive Oxygen Species (ROS) production between <i>piezo</i> mutants and the wild type in an apoplastic ROS assay with a MAMP elicitor (flg22) and also with Ca²⁺ flux leaf disk assay. In conclusion, we demonstrated a potential role for PIEZO in plant immune defense responses and the growth-defense tradeoff.</p>

No description available.

<p>A thesis concerning the effects of water deficit on stomatal traits in plants. The relationships between different traits and their influence on overall stomatal anatomy is discussed. Genetic work to investigate molecular regulation of stomatal development is also presented and discussed.</p>