This research establishes molecular breeding capability for adapting common bean (Phaseolus vulgaris L.) to UK growing conditions. A high-resolution linkage map was constructed for a bi-parental recombinant inbred population (large brown x small white haricot) using genotyping-by-sequencing data. Pre-breeding material was exploited to enable genetic mapping and marker-assisted selection of essential adaptive traits, including (1) resistance to halo blight, caused by Pseudomonas syringae pathovar phaseolicola (Psph), (2) root architecture related to abiotic stress tolerance and nutrient acquisition, (3) earliness of maturity, (4) plant architecture amenable to mechanical harvest, and (5) seed coat colour of consumer interest. A 500-kb mapping interval was defined for quantitative resistance to the broadly virulent Psph race 6, a devastating bacterial pathogen that threatens global bean production with losses from halo blight. Complementary research generating high-quality draft genomes for 32 pathogenically and geographically diverse isolates of Psph identified five high-probability candidate determinants of the broad virulence of Psph race 6, including avirulence protein AvrD. Pathogenicity effectors that are highly conserved within the pathovar were identified as candidate targets for potential race-nonspecific resistance to halo blight. Putative QTL for root architecture traits associated with water and nutrient acquisition were detected on chromosome Pv07. A useful breeding strategy may be to select for larger taproot diameter in view of the comparatively high heritability of this trait. Potentially desirable alleles on Pv07 are linked in coupling phase with the dominant allele of seed coat pigmentation factor P. Identification of lines recombinant for these alleles may prove useful for the introgression of genes governing physiological resilience into white-seeded varieties adapted to UK growing conditions. Provisional QTL for morphological and reproductive traits of agronomic importance, including plant architecture, growth stage and yield, were identified using phenotypic data obtained from pilot field and polytunnel evaluations of the recombinant inbred population.

H. incana is a native species of the Mediterranean and Middle East. As a neophyte (alien) it has undergone a large range expansion in Northern Europe, the Americas, Asia and Australasia. Casual field observations suggested that within its native range, the dominant life strategy of H.incana was annual, whereas in the British flora it was predominantly perennial. Populations from native and alien ranges were studied in the field and in common garden experiments. Phenotypic differences in morphological and physiological characteristics were compared. Plants derived from neophyte British populations made larger leaf rosettes, flowered later (> 140 days) and exhibited a perennial life cycle. Plants from native. North African and Southern European populations (excepting those from montane Spain) made smaller rosettes, flowered early (< 110 days) and died after flowering once. Neophyte populations from California were similar to native populations. Some native populations (e.g. Cypress) did not survive a British winter. Unlike native populations, initiation of flowering in neophyte British populations was stimulated by a period of vernalisation. These results suggest that life strategy changes have occurred in neophyte populations of H. incana as this species expanded its range northwards, and implies possible genetic differences. Ten microsatellite primers, previously described for related Brassicaceae species, were therefore investigated for potential use in the assessment of H. incana population genetic structure. Five primers successfully amplified a product of expected size, of which 3 were subscequently sequenced to confirm the presence of the SSR. The application of real-time PCR DNA melting analysis to identify SSR variation was investigated using Roche SYBR green and Corbett HRM platforms. SSR variation could be detected using DNA melt analysis, but due to difficulty identifying the composition of heterozygous SSR's the technique could not be sufficiently refined to investigate population diversity. However, preliminary results indicated possible SSR variation between isolated populations.

High soil salinity is a major environmental stress that adversely affects crop production throughout the world. It is now estimated that half of the world’s cropland is affected by salt stress. To cope with various environmental stresses, plants are able to spatially and temporally regulate gene expression through changes in DNA methylation and chromatin conformation, known as epigenetic modifications. Recent studies indicated that epigenetic modifications induced by environmental stress can be inherited over several generations, despite a genomewide epigenetic resetting of epigenetic imprints that takes place during plants reproduction. In this thesis, I evaluated in Arabidopsis thaliana the effect of multigeneration salt stress treatments on the genome-wide dynamics of DNA methylation and tolerance to high salinity. My results show that the immediate progenies of stressed plants displayed better germination and survival rate under high salinity, but contrary to current theories this effect is lost in the following non-stressed generation. Genome-wide DNA methylation analysis revealed that stress induced discrete de novo methylation and demethylation changes on epigenetically labile regions of the plant genome. These acquired tolerance and methylation marks are likely under parent-of-origin control as a result of a robust epigenetic reprogramming that takes place in the male germline. Stress-induced methylation marks identified are associated with transcriptional changes of stress responsive genes and correlated with antisense long-non coding RNA expression. Overall this work establish for the first time a link between differential DNA methylation, gene expression and shortterm adaptation to stress in plants.

The objective of this study is to develop a versatile vector system for the delivery and expression of transgenes in the chloroplast genome of N. benthamiana. The successful advent of such a system would vastly streamline the construction process of chloroplast transformation vectors for the expression of recombinant proteins, such as vaccine candidates, in the chloroplasts of N. benthamiana. Transgenes targeted to the chloroplasts of higher plants are expected to be expressed at considerably higher levels as compared to nuclear expression, resulting in more significant accumulation of recombinant proteins. In this study, a 2-part chloroplast transformation vector system was developed and two new GFP vector prototypes, pEXPR-G and pEXPR-UG were generated for preliminary evaluation of functionality. The aadA and GFP expression cassettes of pEXPR-G and pEXPR-UG were evaluated in E. coli prior to actual delivery into N. benthamiana via particle bombardment. Particle bombardment parameters were optimised with particular emphasis on minimising excessive damage to the target tissue in order to facilitate the recovery of antibiotic resistant shoots and calli following transformation. To further evaluate the versatility of the developed system for the expression of vaccine antigens, recombinant vectors, pEXPR-HA and pEXPR-NA were constructed for the delivery of hemagglutinin (HA) and neuraminidase (NA) genes of avian influenza strain H5N1 into the chloroplast genome of N. benthamiana. Experimental results indicated that pEXPR-G and pEXPR-UG were fundamentally functional in E. coli and both the aadA and GFP expression cassettes were active, allowing the bacteria to withstand 500mg/l spectinomycin and express the transgene of interest at the protein level. Similar results were also observed in transplastomic N. benthamiana transformed with pEXPR-UG and pEXPR-NA. In essence, the developed 2-part chloroplast transformation vector system was found to be highly versatile and could be conveniently applied for the construction of transformation vectors for the delivery and expression of HA and NA in the chloroplast of N. benthamiana.

Plants rely on light to supply photosynthetic energy and to provide information of the surrounding environment. Phytochromes are photoreceptors that sense external light quality and quantity, which in turn guide the strategy of plant growth. A large body of research has focused on Arabidopsis thaliana seedlings, where phytochrome control of responses such as hypocotyl elongation, hook opening and cotyledon greening, has been intensively explored. Mathematical models have also helped elucidate the molecular mechanism of phytochrome signalling. A smaller proportion of studies have investigated the role of phytochrome in controlling plant plasticity in adult plants. This work has shown that phytochrome depletion enhances leaf petiole elongation and slows growth, but there is a lack of information on how these marked changes alter metabolism. In this thesis, I use phytochrome multiple mutants of to explore how phytochromes interact with metabolism to affect plant growth. My analysis revealed that phytochrome loss results in dramatically reduced biomass production, especially in high order phyABDE mutant that lacks four out of five phytochromes. This is caused, at least partly, by impaired photosynthesis in phytochrome mutants, including reduced chlorophyll level and less CO2 uptake. Furthermore, cell wall synthesis and protein levels, major dry biomass constituents, are also repressed in phytochrome-depleted plants. Interestingly, these mutants accumulate more daytime sucrose and starch than wild type does, possibly due to their retarded growth in light. Further metabolic profiling reveals that these phytochrome mutants over-accumulate sugars, organic acids and amino acids. The sizable increase in raffinose and proline suggests a possible link to stress tolerance. Indeed, ABA and salt responses are significantly reduced in phytochrome mutants at both seedling and adult stages. These mutants are also more resistant to prolonged darkness, with less chlorophyll degradation in dark and higher survival rates. Collectively, this thesis shows that phytochromes have a novel role in plant resource management, controlling the allocation of resources for growth, switching the metabolism between growth and stress-coping states based on the availability of light from the environment. It brings new interest into phytochrome research in Arabidopsis, suggesting possible application of such knowledge to crop studies in the future.

The chloroplast protein CP12 is present in almost all photosynthetic organisms. This protein has been shown to regulate the activity of two enzymes of the Calvin-Benson cycle, namely glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). The regulation of these enzymes is achieved by the reversible formation of this multiprotein complex in response to a change in light intensity. In Arabidopsis, there are three CP12 genes, CP12-1, CP12-2 and CP12-3. Expression analysis of these genes suggested that they may have a wider role in non-photosynthetic plastids through the plants’ life cycle and that their function may not be restricted to the Calvin-Benson cycle. The main aim of this study was to determine the functional significance of having three CP12 isoforms and to explore the importance of each individual isoform in vivo. This was done by using Arabidopsis thaliana T-DNA mutant and RNAi transgenic lines with a reduced level of CP12. Our results revealed that single mutant lines did not develop a severe growth phenotype. However, a reduction in the transcript of more than one CP12 gene, in a number of multiple lines, led to a significant reduction in photosynthetic capacity at early stages of development and a severe growth phenotype, including reduced fresh and dry weight, number of leaves and seed yield, as well as affected lateral roots formation. Complementation analysis of CP12-1 in the triple mutant revealed that two out of the three lines rescued the phenotype by showing normal growth and development, confirming the importance of CP12. Our results suggest that the CP12 protein family is essential for normal growth and development and that these proteins are likely to have additional functions apart from the regulation of Calvin-Benson cycle enzymes.

The ability to withstand environmental temperature variation is essential for plant survival. Formative studies in Arabidopsis have revealed that light signalling pathways has a potentially unique role in shielding plant growth and development from seasonal and daily fluctuations in temperature. In this thesis we further investigate the integration of the light signalling networks and temperature signalling on the molecular level in Arabidopsis. First, we identified the transcript of the bHLH transcription factor LONG HYPOCOTYL IN FAR-RED 1 (HFR1) to be highly dependent on the ambient temperature and under strong control of the red light photoreceptor PHYTOCHROME B (phyB). We found that the long hypocotyl phenotype of the hfr1 mutant was exaggerated in warm conditions, specifically in blue light, downstream of cryptochrome 1. We further show that HFR1 acts in the warm by suppressing the function of PHYTOCHROME INTERACTING FACTOR 4 and 5 (PIF4, PIF5). PIF4 appears to act as a master regulator of several temperature responses and is directly regulated by the phytochromes. Thus, we define a molecular network where red light and blue light signals together with temperature merge on the regulation of PIF4. In the second part of this thesis, we investigate the relationship between temperature and the fluence rate of light on the inhibition of hypocotyl elongation in Arabidopsis. We find that the response to increasing fluence rates of light is highly dependent on the ambient temperature and that PIF4 and PIF5 acting downstream of the major red light photoreceptor, phyB, are essential for this response. In addition, we provide evidence that in cool conditions, PIF activity is under strong suppression by the gibberellin and HY5 pathways specifically at high fluence rates of red light. The collected work of this thesis highlights the importance of the PIF proteins as integrators of temperature and light signals and furthermore, demonstrates that the response to temperature is highly dependent on both the quality and quantity of light.

Kambiz C. Zangi. / Also issued as Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 1994. / Includes bibliographical references (p. 152-157). / Supported by the U.S. Air Force Office of Scientific Research. AFOSR-91-0034-C Supported by the U.S. Navy Office of Naval Research. N00014-93-1-0686 Supported by Lockheed Sanders, Inc.

In order to examine population structure (micro-evolution) in Begonia and its congruence to higher patterns of diversity (macro-evolution), nuclear microsatellite markers have been isolated and applied to two Begonia species, B. socotrana and B. sutherlandii. Begonia socotrana is endemic to the Haggeher Mountains of the island of Socotra in the Indian Ocean, where it has a total range of less than 10 x 15 km. Population surveys have highlighted the need for its conservation status to be reassessed, and it is proposed to reduce its status from 'endangered' to 'least concern'. Population genetic analyses using microsatellite data show a significant degree of population structure (RST= 0.081, P<0.01; q=0.096, P<0.01) and significant isolation by distance, even over small spatial scales. The pattern of isolation by distance could be due to restricted gene flow, or the result of small scale vicariance events in the fragmented peaks of the Haggeher Mountains during climate change and resulting altitudinal migration. Begonia sutherlandii is native to eastern and southern Africa, where it is restricted to shaded, moist banks in indigenous forest. A high degree of population structure was found (q=0.482, P<0.001; RST=0.634, P<0.001), which along with a high number of private alleles reflects the severe isolation of populations in a patchily distributed forest habitat. Population relationships appear to be strongly governed by the history and continuity of forest cover in the region. The population genetic studies of B. socotrana and B. sutherlandii show a strong correlation of genetic variation with geography which reflect patterns seen at larger scales. The correlation of micro and macro evolutionary patterns is congruent with a hypothesis of restricted gene flow promoting speciation in Begonia.

El propòsit d'aquesta tesi és situar el paper del silenci dins del procés de la comunicació, partint de la convicció inicial que el seu caràcter contradictori amb la parla però a la vegada inherent a ella, necessita un especial anàlisi des d'una teoria general de la comunicació.Paraula i silenci s'expliquen com a conceptes no oposats, sinó necessaris recíprocament. El caràcter fronterer del silenci entre el món de la comunicació verbal i no verbal explica l'òptica interdisciplinar que predomina en aquest estudi, el qual, a partir de pressupòsits de la teoria de la comunicació i de la pragmàtica, especialment, incideix en la seva concepció en diferents espais del saber, des del món de la literatura o la religió al món de les arts, tenint en compte tanmateix qüestions terminològiques o la relativitat cultural a la qual està sotmés.La quantitat de tipologies i funcions atribuïdes al silenci demostra la seva qualitat d'espai operatiu i interioritzat pels parlants; per tant, una sistematització subordinada a l'anàlisi contextual no pot ser aliena al futur de la Lingüística. / El objetivo de esta tesis es situar el lugar del silencio en el proceso de la comunicación, partiendo de la convicción inicial de que su misma naturaleza contradictoria con el habla pero a la vez inherente a ella, necesita un especial análisis desde una teoría general de la comunicación. Palabra y silencio se explican como conceptos no opuestos, sino necesarios recíprocamente. La naturaleza fronteriza del silencio entre el mundo de la comunicación verbal y no verbal, así como su carácter no lineal, heterogéneo y complejo, explica el enfoque interdisciplinar que predomina en esta tesis, el cual partiendo de los presupuestos teóricos propios de la teoría de la comunicación y la pragmática, especialmente, hace hincapié asimismo en su concepción en diferentes espacios del saber, desde el mundo literario o la religión al mundo de las artes, teniendo en cuenta cuestiones terminológicas o la relatividad cultural a la que el silencio está sometido.La creciente cantidad de tipologías y funciones atribuidas al silencio demuestra que es un espacio operativo e interiorizado por los hablantes, por lo que una sistematización, subordinada al análisis contextual, no puede ser ajena al futuro de la Lingüística. / This dissertation strives to ascertain the role of silence in the communication process. The starting point is the conviction that silence's contradictory relationship towards speech, that it both negates and illuminates, requires a special mode of analysis, inserted in a general theory of communication.Both world and silence exist as complementary notions that do not oppose each other. The liminarity of silence ?equidistant to both, verbal and non-verbal communication? requires the interdisciplinary approach that this study uses. Starting from the theoretical principles of both pragmatics and communication theory, it encompasses also the contributions from disciplines as diverse as literature, religion and art. It takes into account issues of terminology, and also the forces of cultural relativism to which it is subject to.The increasing number of functions and tipologies attributed to silence proves its necessity as an operative and interiorized notion among speakers. Its sistematic study, subordinated to contextual analysis, should be an important discipline in the future study of Linguistics.