Engineering morphogenesis of Marchantia polymorpha gemmae

Morphogenesis is an apparent yet complex process: emergence of a plant shape is the result of an intricate interplay between genetic regulation, cell physiology and mechanical processes at the tissue scale. Morphogenesis spans three levels of biological organisation, forming a nested complex system. Genes, which form the lowest level of the system, are arranged into networks that control properties of cells. Cells, which form the middle level of the system, are arranged into geometric networks, and their mechanical and chemical interactions give rise to the morphology of a whole organism. Therefore, the study of plant morphogenesis relies on understanding how genetically-driven cellular interactions influence the formation of a plant shape. Clonal propagules of Marchantia polymorpha (Marchantia), also known as gemmae, are an attractive system for studying these interactions. Gemmae are small, have a simple disk-like shape and are resilient to environmental conditions. As such, they are well-suited for fluorescent microscopy and the collection of gene expression data. These features create an opportunity to study the processes of gemma morphogenesis at tissue, cellular and genetic levels through fluorescent microscopy in a single assay. In order to enable the engineering of morphogenesis in Marchantia gemmae, tools and frameworks for obtaining, storing and analysing the observations from the three levels of biological organisation should be put into place. The work presented in this thesis focuses on the development of such tools and their application in studying the role of phytohormone auxin in Marchantia development. I introduced novel sample preparation and time-lapse imaging assays for Marchantia gemmae along with image- processing methods for the estimation of tissue expansion rates with sub-cellular resolution. These methods allowed me to hypothesise a mechanism behind the regulation of cell proliferation in Marchantia and the role auxin plays in controlling this process. Together with Bernardo Pollak, I developed MarpoDB, a gene-centric representation of the Marchantia genome that enables the search and preparation of Marchantia genetic parts for assembly into synthetic DNA constructs. I used MarpoDB to extract parts and build fluorescent reporters, providing proxies for auxin biosynthesis and signalling. The reporters were then introduced into the Marchantia genome, and the gemmae of the transgenic lines were imaged to estimate the average patterns of the reporters’ expression. The collected patterns were then overlaid on the patterns of relative tissue expansion to validate the proposed role of auxin as an inhibitor of cell proliferation and the mechanism behind its transport in Marchantia.BBSR

UVR8 mediated spatial differences as a prerequisite for UV-B induced inflorescence phototropism

In Arabidopsis hypocotyls, phototropins are the dominant photoreceptors for the positive phototropism response towards unilateral ultraviolet-B (UV-B) radiation. We report a stark contrast of response mechanism with inflorescence stems with a central role for UV RESISTANCE LOCUS 8 (UVR8). The perception of UV-B occurs mainly in the epidermis and cortex with a lesser contribution of the endodermis. Unilateral UV-B exposure does not lead to a spatial difference in UVR8 protein levels but does cause differential UVR8 signal throughout the stem with at the irradiated side 1) increase of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), 2) an associated strong activation of flavonoid biosynthesis genes and flavonoid accumulation, 3) increased GA2oxidase expression, diminished gibberellin1 levels and accumulation of DELLA protein REPRESSOR OF GA1 (RGA) and, 4) increased expression of the auxin transport regulator, PINOID, contributing to local diminished auxin signalling. Our molecular findings are in support of the Blaauw theory (1919), suggesting that differential growth occurs trough unilateral photomorphogenic growth inhibition. Together the data indicate phototropin independent inflorescence phototropism through multiple locally UVR8-regulated hormone pathways

Tree Peony Species Are a Novel Resource for Production of α-Linolenic Acid

Tree peony is known worldwide for its excellent ornamental and medical values, but recent reports that their seeds contain over 40% α-linolenic acid (ALA), an essential fatty acid for humans drew additional interest of biochemists. To understand the key factors that contribute to this rich accumulation of ALA, we carried out a comprehensive study of oil accumulation in developing seeds of nine wild tree peony species. The fatty acid content and composition was highly variable among the nine species; however, we selected a high- (P. rockii) and low-oil (P. lutea) accumulating species for a comparative transcriptome analysis. Similar to other oilseed transcriptomic studies, upregulation of select genes involved in plastidial fatty acid synthesis, and acyl editing, desaturation and triacylglycerol assembly in the endoplasmic reticulum was noted in seeds of P. rockii relative to P. lutea. Also, in association with the ALA content, transcript levels for fatty acid desaturases (SAD, FAD2 and FAD3), which encode for enzymes necessary for polyunsaturated fatty acid synthesis were higher in P. rockii compared to P. lutea. We further showed that the overexpression of PrFAD2 and PrFAD3 in Arabidopsis increased linoleic and α-linolenic acid content, respectively and modulated their final ratio in the seed oil. In conclusion, we identified the key steps that contribute to efficient ALA synthesis and validated the necessary desaturases in P. rockii that are responsible for not only increasing oil content but also modulating 18:2/18:3 ratio in seeds. Together, these results will aid to improve essential fatty acid content in seeds of tree peonies and other crops of agronomic interest

Characterizing biological systems: quantitative methods for synthetic genetic circuits in plants and intracellular mechanics

2018 Summer.Includes bibliographical references.To view the abstract, please see the full text of the document

Discrete Event Simulations

Considered by many authors as a technique for modelling stochastic, dynamic and discretely evolving systems, this technique has gained widespread acceptance among the practitioners who want to represent and improve complex systems. Since DES is a technique applied in incredibly different areas, this book reflects many different points of view about DES, thus, all authors describe how it is understood and applied within their context of work, providing an extensive understanding of what DES is. It can be said that the name of the book itself reflects the plurality that these points of view represent. The book embraces a number of topics covering theory, methods and applications to a wide range of sectors and problem areas that have been categorised into five groups. As well as the previously explained variety of points of view concerning DES, there is one additional thing to remark about this book: its richness when talking about actual data or actual data based analysis. When most academic areas are lacking application cases, roughly the half part of the chapters included in this book deal with actual problems or at least are based on actual data. Thus, the editor firmly believes that this book will be interesting for both beginners and practitioners in the area of DES

Discovering Regularity in Point Clouds of Urban Scenes

Despite the apparent chaos of the urban environment, cities are actually replete with regularity. From the grid of streets laid out over the earth, to the lattice of windows thrown up into the sky, periodic regularity abounds in the urban scene. Just as salient, though less uniform, are the self-similar branching patterns of trees and vegetation that line streets and fill parks. We propose novel methods for discovering these regularities in 3D range scans acquired by a time-of-flight laser sensor. The applications of this regularity information are broad, and we present two original algorithms. The first exploits the efficiency of the Fourier transform for the real-time detection of periodicity in building facades. Periodic regularity is discovered online by doing a plane sweep across the scene and analyzing the frequency space of each column in the sweep. The simplicity and online nature of this algorithm allow it to be embedded in scanner hardware, making periodicity detection a built-in feature of future 3D cameras. We demonstrate the usefulness of periodicity in view registration, compression, segmentation, and facade reconstruction. The second algorithm leverages the hierarchical decomposition and locality in space of the wavelet transform to find stochastic parameters for procedural models that succinctly describe vegetation. These procedural models facilitate the generation of virtual worlds for architecture, gaming, and augmented reality. The self-similarity of vegetation can be inferred using multi-resolution analysis to discover the underlying branching patterns. We present a unified framework of these tools, enabling the modeling, transmission, and compression of high-resolution, accurate, and immersive 3D images

Computational Morphodynamics: A Modeling Framework to Understand Plant Growth

Computational morphodynamics utilizes computer modeling to understand the development of living organisms over space and time. Results from biological experiments are used to construct accurate and predictive models of growth. These models are then used to make novel predictions that provide further insight into the processes involved, which can be tested experimentally to either confirm or rule out the validity of the computational models. This review highlights two fundamental challenges: (a) to understand the feedback between mechanics of growth and chemical or molecular signaling, and (b) to design models that span and integrate single cell behavior with tissue development. We review different approaches to model plant growth and discuss a variety of model types that can be implemented to demonstrate how the interplay between computational modeling and experimentation can be used to explore the morphodynamics of plant development

GETTING TO THE ROOT CAUSE: THE GENETIC UNDERPINNINGS OF ROOT SYSTEM ARCHITECTURE AND RHIZODEPOSITION IN SORGHUM

Plants are some of the most diverse organisms on earth, consisting of more than 350,000 different species. To understand the underlying processes that contributed to plant diversification, it is fundamental to identify the genetic and genomic components that facilitated various adaptations over evolutionary history. Most studies to date have focused on the underlying controls of above-ground traits such as grain and vegetation; however, little is known about the “hidden half” of plants. Root systems comprise half of the total plant structure and provide vital functions such as anchorage, resource acquisition, and storage of energy reserves. The execution of these key functions via root system architecture and root exudation directly determines plant performance, and thus reproductive fitness. Despite the significance of roots, the genetic controls contributing to their variation have gone understudied due to the technical difficulties associated with below-ground phenotyping. Domesticated plants provide an excellent framework for studying the genomic underpinnings of phenotypic diversity due to the telescoped evolutionary time frame under which artificial selection took place. The rapid evolution of domesticates from their extant antecedent affords direct observations of derived traits and their underlying genetic controls. Sorghum, a globally important domesticated grass species with a small diploid genome, few genetic repeats, and a wide variety of adaptations, serves as a good model for studying selection during domestication. Domesticated S. bicolor is an annual accession with large seeds and flowering organs compared to its wild relative, S. propinquum. Comparatively, S. propinquum is perennial with dense rhizomes and small flowering panicles. Due to their distinctly opposing root systems, a recombinant inbred line (RIL) population formed from a cross between S. bicolor and S. propinquum was used to identify the specific root morphological and metabolic adaptations that derived from domestication. The RIL population was phenotyped using high-throughput image analysis to locate the underlying genomic factors controlling derived traits via high-density Quantitative Trait Loci (QTL) mapping. Nine novel QTL influencing root morphology were identified. No QTL were identified for metabolic exudation; however, crown root growing angle was found to be a statistically significant predictor of the percentage of carbon and nitrogen in the rhizosphere. The relationship between steep growing angles and increased rhizosphere carbon and nitrogen suggests that increased exudation was derived during domestication. Candidate genes and pathways were identified including those that encode meristem transcription factors, plant hormone receptors, and actin trafficking. These findings advance our understanding of the underlying genomic factors controlling root system architecture (RSA) and root exudation that were selected during the domestication of Sorghum. The results of this study can be integrated into breeding programs for the establishment of elite root lines used to mitigate the effects of current and future environmental challenges of croplands in a sustainable manner