Plant seeds progress through specific stages during germination, from quiescence in the dry state through water uptake, testa rupture and finally endosperm rupture. The stages of seed germination are fairly well classified but the underlying biochemical and mechanical processes are unknown. The ability to control a seeds progression through the stages of germination has implications on farming efficiency and so the following thesis explores Arabidopsis thaliana and Lepidium sativum seeds during the germination process. A systematic approach to analysing the shape of cells within the radicle (embryonic root tissue) is developed, using confocal imaging, in order to characterise the shape of cells in the different tissues of the radicle. The cell shape approximations are not refined enough to characterise the different cell tissues. With more data, this approach would hope to find the region in which cells alter through the germination process. Change in the activity of cell wall modifying enzymes within the endosperm, that surrounds the emerging embryo, is a key part of the germination process and temporally and spatially defined high resolution transcriptomics data-sets are available to inform models. Through the course of this thesis, biochemical networks are developed, with ordinary and partial differential equation models being constructed and analysed. The models highlight elements for further investigation as well as differences between the two species considered. The mathematical models, along with data from biomechanical experiments on the endosperm, inform discussion on how the cell wall biochemistry of a cell wall alters the cell wall properties. These discussions focus on cell wall permeability, extensibility and the final cell separation event associated with germination. From the considered proteins, polygalacturonase and pectin lyase arise as the only viable candidates to cause the cell separation event with the model framework.

The formation of lateral roots is an important post-embryonic developmental process that allows plants to adapt to their environment via exploitation of soil mineral resources. New lateral roots initiate as lateral root primordia (LRP) in the pericycle cell layer adjacent to the central vascular tissue in the primary root, and must pass through the outer cell layers of endodermis, cortex and epidermis to emerge as mature roots. A key regulator of emergence is the plant hormone auxin and it has been shown previously that in Arabidopsis the auxin induced expression of the auxin influx carrier LAX3 in specific cortical cells over LRP is required for emergence to occur, as this leads to the expression of cell wall remodelling enzymes such as polygalacturonase (PG). By developing mathematical models of auxin transport and LAX3 expression the work in the thesis aims to test the existing conceptual models for lateral root emergence, and provide testable hypotheses for the existence of additional gene regulatory components. An initial single cell model demonstrates that hysteresis and bistability may explain the experimentally observed 'all-or-nothing' LAX3 spatial expression pattern in cortical cells containing a gradient of auxin concentrations. By fitting model parameters against experimental data, the model is then used to show that some auxin homeostasis mechanism is present, with both endogenous and exogenous sources of homeostasis investigated. The single cell model also investigates the validity of several alternative gene regulatory networks for LAX3, and its apparent repression by a key mediator of the auxin response, ARF19. Finally, the model is extended to a multicellular context, in which the auxin distribution from a simulated LRP source cell is used as a basis for the expression of LAX3, leading to the expression of PG in specific cells between which the LRP must pass.

The ornamental industry has become an important economic force in recent years, in the UK alone this industry is estimated to be around £2.1 billion, while the international trade is around £60-75 billion (Chandler and Tanaka, 2007). The continued success of the floriculture industry depends on the introduction of new species/cultivars with major alterations in key agronomic characteristics, such as resistance to pathogens, novel flower colour and patterns or control of male fertility. Lilium, one of the most important bulbous ornamental crops, is an attractive and popular cut flower. However, the production of vast quantities of pollen that stains easily and is toxic to animals is not always desirable. The control of pollen release without affecting the appearance of the flower is therefore an important breeding goal. Lilium is also susceptible to several fungal pathogens, including Botrytis cinerea, which infects leaves, stem and flowers leading to a reduction of yield. New cultivars have tended to rely upon selective breeding as a mechanism for trait development. However approaches that utilise transgenes to manipulate traits of interest provide alternative opportunities for the ornamental industry provided that transformation and regeneration can be achieved efficiently. A rapid, highly efficient and reproducible Agrobacterium-mediated transformation for Lilium has been developed. Successful transient GUS expression in callus, shoots and basal plate discs was achieved using A. tumefaciens strain AGL1 containing plasmid pBI121 harbouring intron-containing GUS and NPTII genes in cultivars "Beverly's Dream", "Star Gazer", "Night Flyer", "Acapulco", "Sweet Surrender" and Lilium leichtlinii. Based on the same transformation protocol, transgenic plants of cv. "Star Gazer" overexpressing the RCH10 chitinase gene from rice were generated. In vitro sporulation assays of these plants showed different levels of resistance to Botrytis cinerea correlated to the level of relative expression of the transgene. This is the first report of induced pathogen resistance in any Lilium cultivar by transgenic approach. Experiments were also conducted to modify fertility and pollen release in Lilium by translating regulatory gene information from Arabidopsis to Lilium. Transgenic plants of cv. "Star Gazer" either overexpressing or silencing the AtMYB26 gene, were generated. RNAi lines showed a delay in anther dehiscence suggesting that pollen development pathways could be conserved between Arabidopsis and Lilium. In addition, partial sequences of the putative orthologues of AtMS1 and AtMYB26 in Lilium were identified and cloned for future research.

The control of pollen viability and release is of major commercial importance in the development of crops for hybrid seed production and selective breeding. It has been shown that key transcription factors in Arabidopsis particularly MALE STERILITY1 (MS1), are functionally conserved in rice (Li et al., 2011), therefore extending this comparative analysis and controlling fertility in temperate cereals, such as barley, is the long term goal of this project. Although anther and pollen development of barley seems morphologically similar to Arabidopsis, the genes involved and how they are regulated are currently unknown. Arabidopsis MS1 is a tapetum specific transcription factor which is expressed exclusively from the tetrad stage to early microspores release. Identification and accurate staging of barley anther development is essential for expression analysis and functional characterisation of genes involved in pollen development. Therefore, a complete morphological study of barley development was conducted. External characteristics have been described in parallel to anther development in order to predict anther stages by the observation of external stages phenotypic traits. Characterization of the barley orthologue of MS1 (HvMS1) has been conducted. Recently a new grass genome has been released, Brachypodium distachyion. This new resource has been used to aid primers design alongside the rice OsPTC1 sequence, the orthologue of MS1 (Li et al., 2011). Genome sequencing has indicated that the Brachypodium genus is more closely related to wheat and barley than it is to rice, Due to the close relationship between Brachypodium and barley, this new grass has been used as intermediary to identify the OsPTC1 orthologue in barley as well as downstream MS1 targets. A highly similar sequence to OsPTC1 was found in Brachypodium, Bradi4g31760. This new gene, as a result of its similarities to OsPTC1, was considered as its putative orthologue gene in Brachypodium. Therefore, the most conserved areas between OsPTC1-Bradi4g31760 were used for primers design to successfully amplify equivalent gene in barley (HvMS1). The characterization of this barley gene showed a similar expression pattern to the MS1 putative orthologue in Arabidopsis of tapetum specific expression. In addition, RNAi silencing of this gene has revealed that it is essential for the normal development of pollen, with a lack of viable pollen produced in the putative HvMS1 silenced transgenic lines.

Soil compaction has been described as the most serious environmental problem caused by conventional agriculture, as it results in several stresses which may interact simultaneously, including increased soil strength, decreased aeration and reduced hydraulic conductivity. Root system architecture (RSA) is the arrangement of roots within the soil matrix and is important because the specific deployment of roots within the soil can determine soil exploration and resource uptake. As roots deliver water and nutrients to growing plants, whilst also providing anchorage, their importance cannot be overstated. Yet, our understanding of how roots interact with the surrounding soil, especially at the micro-scale level, remains limited because soil is an opaque medium, so preventing roots from being visualised without disturbing them. Destructive techniques are commonly employed for the analysis of RSA, however this can result in the loss of key information concerning root architecture, such as elongation rates and root angles and important soil characteristics such as soil structure and pore connectivity. However, X-ray Computed Tomography (CT) has been shown to be a promising technique for visualising RSA in an undisturbed manner. The species considered in this thesis were wheat (Triticum aestivum L.) and tomato (Solanum lycopersicum L.). Further information regarding the response of roots to soil compaction has been achieved through the use of X-ray CT, automatic root tracing software and novel image analysis procedures. Soil compaction significantly affected root length, volume, surface area, angle, diameter, elongation rates and root path tortuosity, however the influence of soil texture on root responses to soil compaction was significant. Moderate compaction benefits root growth in clay soil, possibly due to the greater nutrient and water holding capacity, but adversely affected root growth in loamy sand. The results suggest that there is an optimum level of soil compaction for the different soil types. Roots elongated rapidly between 2-3 days after germination (DAG), it is hypothesised that is related to the mobilization of seed storage substances to the growing roots. The use of transgenic mutants of tomato with altered levels of abscisic acid (ABA) has provided a greater insight into the role of ABA in mediating root responses to soil compaction. This work will enable better phenotyping of plant varieties with enhanced root system traits for resource foraging and uptake. Knowledge of the responses of root systems in heterogeneous soil is vital to validate root phenotypes and overcome future food security challenges.

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene, encodes a plant homeodomain (PHD) transcription factor critical for viable pollen formation (Wilson et al., 2001). In the ms1 mutant, there are alterations in the production of pollen wall materials, as well as a failure of tapetal programmed cell death (PCD) (Vizcay-Barrena and Wilson, 2006). This ultimately results in the failure to produce viable pollen. Large numbers of genes are down-regulated in the ms1 mutant indicating that MS1 plays a key role in regulating late tapetal expression and pollen wall deposition (Ito et al., 2007; Yang et al., 2007). Two putative MS1 interacting proteins At1g58210/NET2A (termed as Y2H54) and AT2G46260/ LRB1 (termed as POB2) were identified from a previous Arabidopsis stamen specific yeast-2-hybrid screen, using a truncated version of the MS1 protein without the PHD motif. POB2 and MS1 were found co-localised in the nucleus, while Y2H54 was specifically located at the plasma membrane. Further confirmation of the interaction using Förster resonance energy transfer (FRET) assay methods showed that POB2 failed to interact with MS1 in planta, however, the association between the two proteins occurred in vitro, as confirmed by protein pull-down assays. Additionally, enhanced general plant growth and floral development were seen in the overexpression lines of Y2H54. However, no significant phenotypes were observed in the RNAi silencing lines. Chromatin Immunoprecipitation (ChIP) analysis uncovered that MS1 directly regulated the expression of MYB DOMAIN PROTEIN 99 (MYB99) by binding to its promoter. Other putative MS1 direct targets identified by ChIP include 3-KETOACYL-COA SYNTHASE 7 (KCS7), 3-KETOACYL-COA SYNTHASE 15 (KCS15), SPERMIDINE HYDROXYCINNAMOYL TRANSFERASE (SHT) and TAPETUM-SPECIFIC METHYLTRANSFERASE 1 (TSM1). Histone extraction and western blotting assays suggest a role for MS1 in facilitating detrimethylation of H3 marks. H3K36me3 deposition was enhanced at MYB99 in ms1 compared with the wild type, suggesting that MS1 may regulate MYB99 via H3K36me3. A new model for the MS1 regulatory network in pollen wall formation has therefore been proposed.

Wheat is a crop of global importance accounting for 20% of global calorie consumption and a similar percentage of the daily protein for 2.5 billion people in less developed countries. To meet the food production demands of a predicted global population of 9 billion people by 2050, wheat yields need to increase by 1.7% per annum, whilst facing the added pressures of reduced fertilizer inputs and potentially reduced land availability. Plant root systems are key for efficient water and nutrient uptake and thus have a direct impact on yield, and yet there has been competitively little research into root system improvement. A high-throughput root phenotyping pipeline for wheat seedlings was designed consisting of a germination paper-based growth system combined with image segmentation and analysis software. A number of lines from the A.E. Watkins landrace population were characterised to test the final pipeline design. The pipeline was then utilized to phenotype a population of 94 lines from a doubled haploid population for quantitative trait loci (QTL) discovery. In total, 29 root QTL were discovered, with 2 loci co-localising with QTL for grain yield and nitrogen uptake efficiency discovered in field trials. Modern wheat varieties may have limited genetic diversity, due to changes in ploidy throughout evolution and subsequent domestication. With this in mind, thirty five ancient wheat relatives and eighteen amphidiploid hybrids were phenotyped for seedling root architectural traits, to determine the amount of phenotypic variation within ancient wheat species, and whether this variation can be transferred to modern varieties. The utilization of seedling root trait phenotyping is discussed and future research directions are identified.

Orientadora: Vera Lúcia Scatena / Banca: Maria das Graças Sajo / Banca: Adelita Aparecida Sartori Paoli / Banca: Silvia Rodrigues Machado / Banca: Hildeberto Caldas de Sousa / Resumo: Dentre as Eriocaulaceae (sempre-vivas) brasileiras, alguns representantes de Comanthera e Syngonanthus são os mais comercializados e utilizados na ornamentação e constam da lista brasileira de espécies ameaçadas de extinção. Essas plantas crescem nos campos rupestres, sob situações climáticas adversas. Estudos taxonômicos e filogenéticos de Comanthera subg. Comanthera indicam falta de dados para maior suporte ao subgênero e nomeiam clados na distribuição das espécies. Procurando auxiliar a taxonomia e filogenia do grupo, bem como interpretar as adaptações ao ambiente e fornecer dados para futuro manejo, estudou-se aspectos da morfologia e anatomia de 24 espécies de Comanthera (envolvendo as espécies do clado "V", "U" e "X") e de Syngonanthus nitens. As espécies do clado "V": Comanthera brasiliana, C. brunnea, C. caespitosa, C. magnifica e C. suberosa apresentam germinação e desenvolvimento pós-seminal mais rápido (< 7 dias) do que as demais Eriocaulaceae, e crescem em solo quartzítico pobre em nutrientes, cuja água escoa rápido. Apresentam envoltório seminal com projeções da endotesta com formas diferentes que são úteis para separar as espécies. O levantamento de estruturas anatômicas de folhas e escapos dessas espécies como: tipo de câmara subestomática, estrutura da margem foliar e número de feixes vasculares indicam agrupamentos entre elas e servirão para a sustentação do clado filogenético em que estão inseridas. Estruturas anatômicas como: presença de células de paredes espessadas, de hipoderme e parênquima clorofiliano compacto são respostas adaptativas à escassez hídrica, ventos fortes e radiação excessiva, presentes nos campos rupestres. As espécies de Comanthera subg. Comanthera que ocorrem na Bahia (nove espécies - clado "X") e Minas Gerais (10 espécies - clado "U") apresentam ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Among the Brazilian Eriocaulaceae (sempre-vivas), the species most widely commercialized and used for decoration belong to Comanthera and Syngonanthus, and are already on the Brazilian list of endangered species. These plants grow in upland "campo rupestre" vegetation, under adverse climatic conditions. Taxonomic and phylogenetic studies of Comanthera subg. Comanthera have revealed a lack support for the subgenus and have used clades instead for discussing species distributions. The present study aimed to contribute to the taxonomy and phylogeny of the group, clarify environmental adaptations and provide data for future environmental management of natural populations. Morphological and anatomical features of 24 species of Comanthera (involving species of clades "V", "U" and "X") and of Syngonanthus nitens were studied. Species of clade "V"  Comanthera brasiliana, C. brunnea, C. caespitosa, C. magnifica and C. suberosa  exhibit more rapid germination and post-seminal development (< 7 days) than other Eriocaulaceae, and grow in nutrient-poor quartzite soils with rapid water drainage. They have seed coats with endotestal projections of differing shapes that are useful for separating the species. The survey of anatomical structures of the leaves and scapes of these species, such as the type of substomatal chamber, leaf margin structure and number of vascular bundles, revealed groupings that will serve to support the phylogenetic clade in which they are included. Anatomical structures such as presence of thick cell walls, hypodermis and compact chlorenchyma are important adaptive responses to the water scarcity, strong winds, and excessive radiation prevalent in "campo rupestre" habitats. The species of Comanthera subg. Comanthera that occur in Bahia (nine species - clade "X") and Minas Gerais (10 species - clade "U"), ... (Complete abstract click electronic access below) / Doutor

It has been demonstrated that bakers' yeast reduction of 1-tert-butyl-2-methyl 3-oxo-piperidine-1,2-dicarboxylate gives (2R, 3S), 1-tert-butyl-2-methyl 3-hydroxy-piperidine-1,2-dicarboxylate in 80% chemical yield with >99% d.e. and >97% e.e. Also bakers' yeast reduction of 1-tert-butyl-3-ethyl 4-oxo-piperidine-1,3-dicarboxylate gives (3R, 4S), 1-tert-butyl-3-ethyl4-hydroxy-piperidine-1,3-dicarboxylate in 74% chemical yield with >99% d.e. and >93% e.e. The optical purity and absolute configurations of the hydroxy-ester derivatives were determined by conversion into the corresponding chiral bis-tosylate derivatives of 2- and 3-piperidinemethanol respectively. It has also been shown that bakers' yeast reduction of 1-tert-butyl-4-methyl 3-oxo-piperidine-1,4-dicarboxylate gives (3R, 4R)-1-tert-butyl-4-methyl 3-hydroxypiperidine-dicarboxylate in 81% chemical yield with >99% d.e. and 87% e.e. The optical purity and absolute configuration of the hydroxy-ester derivative were determined by utilisation of the compound in the total synthesis of (R)-3-quinuclidinol via chain elongation at C-4 of the piperidine followed by cyclisation to produce the bicyclic structure. Further work is reported on the diastereoselective synthesis of polyhydroxylated indolizidine alkaloids. 1-Acetoxy-2-hydroxy-3-(hydroxymethyl)-indolizidine has been synthesised as a single diastereomer from 2-piperidinemethanol via attack of an amine onto an epoxide functionality thus producing the bicyclic system.

Thesis (doctoral)--Universität Zürich, 1909. / Cover title. Includes bibliographical references.