The model angiosperm Arabidopsis thaliana produces viable seed through coordinated growth of three constituents; seed coat, embryo and endosperm. During development the embryo grows to fill the space defined by the seed coat. The growing embryo is surrounded by endosperm, an ephemeral, nutritive structure. The process of programmed cell death (PCD) is essential for endosperm consumption by the embryo however very little is known about developmental PCD in the endosperm. ZOU is a transcription factor expressed specifically in endosperm adjacent to the growing embryo in the Embryo Surrounding Region (ESR) (Yang et al., 2008). zou seed likely have reduced PCD resulting in abnormally persistent endosperm and a small embryo at seed maturity which results in seed shriveling. A second zou phenotype is an impairment of cuticle development in the embryonic leaves (cotyledons), suggesting that ZOU may mediate a signal from endosperm to embryo. The ESR expressed gene ALE1 is down-regulated in zou. When ALE1 is artificially expressed in zou ESR by the construct pSUC5::ALE1 the zou epidermal phenotype is rescued but not the seed shriveling phenotype of zou (Xing et al., 2013). Fixed and resin-embedded sections of zou and pSUC5::ALE1 lines herein confirm that zou-like endosperm is exhibited in pSUC5::ALE1 lines. This confirms that the two phenotypes of zou are genetically separable. The involvement of ZOU in epidermal processes is further confirmed through genetic studies showing that ZOU acts in the same pathway to impart embryonic cuticle as the embryo-expressed Receptor Like Kinases GSO1 and GSO2. In order to quantify PCD in the endosperm of wild-type and zou seed, PCD expression marker and TUNEL analysis were conducted. One PCD marker, pCEP:H2A-YFP is shown to be expressed in wild-type ESR, it is not clear if expression is lost in zou. To identify candidate genes under the control of ZOU active in endosperm PCD the results from several transcriptional profiling experiments were analysed and validated; this detailed gene expression in wild-type, ale1 and zou siliques which allowed for the identification of targets of ZOU but not of ALE1, targets predicted to be PCD effectors. In silico expression and ontology analysis confirmed likely roles for some candidates in endosperm PCD processes (particularly cell wall modification). Selected targets were cloned under pSUC5 and expressed in the ESR of zou seed as part of a molecular screen for the rescue of the zou endosperm phenotype. The ZOU target FRINGE-Like, a Glycosyl Transferase which shows strong endosperm expression is shown to partially rescue the zou phenotype but does not rescue the epidermal phenotype, suggesting that it may mediate PCD processes under ZOU control. The initial discovery that a Glycosyl Transferase may be active in a developmental PCD process in plants is exciting and novel and benefits understanding of developmental PCD and endosperm breakdown, two poorly characterized processes in plants.

The ‘epigenome’ refers to a difference in the transcriptional behaviour of a given allele that cannot be explained by differences in the genetic code, or ‘mutation’ at that locus. The epigenome is associated with certain biochemical marks, and generally exerts a silencing effect upon the transcription of genes under its influence. In Angiosperms such as Arabidopsis thaliana, fertilisation of both the egg and central cell– referred to as ‘double fertilisation’ – giving rise to the embryo and endosperm respectively, the latter of which is one of the most important tissues in the human food chain. Upon double fertilisation the gametes of each parent are known to contribute differing epigenetic ‘imprints’, where one gamete contributes a copy of a given allele in a transcriptionally unavailable state, while the other parent’s copy is in an available state. When a gene resides in such a region, the result is that only one parent’s copy is hence transcribed; such a gene is said to be ‘imprinted.’ Imprinting is known to affect the development of the placenta as well as some of the adult tissues in mammalian models, and in plants is most extensively found in the endosperm, where without the imprint of both parents the resulting seed exhibits reduced viability and defective endosperm development. A relative dearth in the number of known imprinted loci in the model angiosperm, A. thaliana makes is difficult to make reliable assessments of its role and regulation. This thesis initially aimed to extend the count of known imprinted genes using a model that proved insufficient to identify novel imprinted genes, and presents a meta-analysis showing that the reliable attribution of imprinted status to a gene is difficult using high-throughput methods as well. In addition, the further characterisation of a novel imprinted gene identified previously by this lab, MPC, with a view to acquiring a more detailed understanding of its role using mutants carrying point mutations in the MPC protein showed only a subtle phenotype to discern them from wild-type plants. There has also been recent speculation of a role for repeat elements in imprinting. This thesis presents findings suggesting that the apparent association of repeat elements with imprinted genes is an artefact rather of an association of endosperm-expressed genes with transposable elements, rather than genes that are specifically imprinted.

Made available in DSpace on 2014-06-11T19:22:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-03-01Bitstream added on 2014-06-13T18:48:45Z : No. of bitstreams: 1 rossi_rf_me_botfca.pdf: 365226 bytes, checksum: 61d4bb5c47783421434379c08c1d49bb (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A cultura da soja, em razão de sua importância no agronegócio mundial, é alvo de inúmeras pesquisas no campo fitotécnico na busca por novas informações para elevação da produtividade. O vigor é um dos principais atributos da qualidade fisiológica das sementes a ser considerado na implantação de uma lavoura, pela influência que pode ter no crescimento e no rendimento das plantas. A pesquisa teve como objetivo avaliar o desempenho de plantas de soja e a qualidade das sementes produzidas em diferentes densidades populacionais, em função do nível de vigor das sementes utilizadas. O experimento foi conduzido em duas etapas. A primeira constituiu da fase de campo, mediante delineamento em bloco ao acaso, com quatro repetições, em esquema fatorial 3x3 (densidades populacionais de 7, 12 e 17 pl.m-1 e níveis de vigor das sementes alto, médio e baixo), empregando-se três cultivares de soja. Foram avaliados a população inicial, a data do florescimento, o ciclo, a altura média das plantas e da inserção da primeira vagem, os números médio de vagens por planta e de sementes por vagem, a população final de plantas, em relação à inicial, e a produção de sementes. A segunda etapa foi realizada no laboratório e constou da avaliação das características qualitativas das sementes de soja produzidas na etapa anterior. Foi evidente o efeito do vigor das sementes na emergência de plântulas em campo, sendo superior quando oriundas das sementes de alto vigor. A elevação da densidade de plantas de soja na linha ressalta, positivamente, efeitos dos níveis de vigor no desenvolvimento das plantas. O vigor das sementes e a densidade de plantas na linha têm influencia na produção de soja; elevações de 8% e 6% na produção foram observadas em plantas originadas de sementes com vigor médio e alto, em relação às... / Due to the importance of soybean in the world agribusiness, this crop has been the subject of innumerous research studies, looking for management techniques to increase production per area. The vigor is one of the main seed physiological quality characters that has to be considered in the crop establishment, because it can influence the plant growth and final production. This study aimed to evaluate the performance of soybean plants and the quality of seeds produced in different population densities, depending on the level of vigor of the seed sown. The experiment was conducted in two stages. The first one was conducted at the field, in randomized block design, with four replications, in a factorial 3x3 design (densities of 7, 12 and 17 pl.m-1 and levels of seed vigor, high, medium and low), using three soybean cultivars. The following parameters were: initial population, flowering cycle, average height of plants and first pod; average number of pods per plant and seeds per pod; final population of plants (in relation to the initial population); and seed yield. In the second stage, conducted in the laboratory, the quality of the produced seed was evaluated. The effect of seed vigor on seedling emergence in the field was evident, being higher when derived from the seeds of high vigor. The high density of soybean plants in the row highlights positively the effects of vigor levels upon plants development. Seed vigor and plant density on the line have influence on soybean production; increases of 8% and 6% in seed yield were observed in plants from seeds with medium and high vigor, compared to those coming from low vigor. Seed vigor and plant density on the line do not affect the physiological quality of the produced seeds

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination and post-germination. Here I test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating and post-germination seeds. It is demonstrated that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean seed germination and post-germination. Immunogold electron microscopy (EM) with VSR antibodies demonstrates that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in Day-1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at Day-3 post-germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating and post-germination seeds. Further immunogold EM studies demonstrate that VSR and aleurain colocalize to MVBs, as well as PSVs in germinating and post-germination seeds. Thus, MVBs in germinating and post-germination seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed, and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination and post-germination. / Storage proteins synthesized during seed development are transported to PSVs for storage. However, relatively little is known about the mechanisms of storage protein transport. A putative VSR-interacting protein termed S2 was identified as mung bean 8S globulin. Thus, I test the hypothesis that VSR proteins may be involved in storage protein transport to PSVs in developing mung bean seeds. Immunogold EM with 52 (8S globulin) antibody demonstrates that transport of 8S globulin to PSVs is Golgi-mediated, involving dense vesicle (DV) and a novel prevacuolar compartment (PVC). The novel PVC consists of storage protein aggregates and small internal vesicles. Immunogold EM with S2 (8S globulin) antibody demonstrates that MVBs contain 8S globulin at early stage of seed development. Further immunogold EM studies demonstrate that VSR and 8S globulin colocalize to DVs and the novel PVCs. In vitro binding study demonstrates that calcium ion can stabilize interaction between VSRs and 8S globulin. Thus, VSR proteins may mediate storage protein transport to PSVs via a novel PVC. / Wang, Junqi. / "March 2007." / Adviser: Jiang Liwen. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0052. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 120-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Made available in DSpace on 2014-06-11T19:30:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-29Bitstream added on 2014-06-13T18:41:19Z : No. of bitstreams: 1 cazetta_e_dr_rcla.pdf: 968988 bytes, checksum: 4bdfab726fe1de13903afa950d3cf54e (MD5) / Universidade Estadual Paulista (UNESP) / As características dos frutos como morfologia e química têm sido tradicionalmente explicadas como resultado da seleção dos dispersores de sementes. Mas a importância dos frugívoros em moldar as características dos frutos tem sido recentemente questionada sob diferentes perspectivas. Alguns estudos sugerem que outras interações podem simultaneamente atuar sobre a evolução das características dos frutos. Muitos organismos atuam como predadores de sementes e seus efeitos precisam ser considerados. Desta maneira, as plantas enfrentam um dilema evolutivo entre atração aos legítimos dispersores de sementes e defesa contra predadores e patógenos. Neste estudo, primeiramente nós avaliamos as características dos frutos relacionadas com atração e defesa em uma ampla amostra de frutos da Mata Atlântica, da Ilha do Cardoso, São Paulo, Brasil. Posteriormente, nós testamos como algumas características específicas influenciam o consumo pelas aves frugívoras. Nós observamos que as características morfológicas e químicas dos frutos apresentam sinal filogenético e em geral padrões independentes de co-variação. Ao contrário, as cores e os contrates dos frutos não apresentam sinal filogenético e frutos mais saturados são ricos em lipídeos e energia mas pobres em carboidratos. Portanto, nós sugerimos que o grau de saturação da cor pode indicar a qualidade nutricional dos frutos. As aves detectaram consistentemente frutos com maior contraste cromático do que o contraste acromático. Além disso, as aves frugívoras selecionam frutos ricos em lipídeos e energia e pobres em compostos secundários. Nós concluímos que a preferência das aves por determinadas características dos frutos não afeta necessariamente a evolução das mesmas, uma vez que, para isso ocorrer é necessário que as aves selecionem entre indivíduos... / Fruit characteristics such as morphology and chemistry have traditionally been explained as the result of adaptations to their seed dispersers. But the importance of frugivores in shaping fruit traits has now been questioned from a number of perspectives. Some studies suggested that other interactions simultaneously shape the evolution of fruit traits. Many organisms act as seed predators and their effects must be taken into account. Therefore, plants are faced with an evolutionary dilemma between attraction to legitimate seed dispersers and defense against seed predators and pathogens. In this study we focused on fruit traits related to attractiveness and defense. We first evaluated how fruit characteristics interact in a broad sample of Atlantic rainforest species, at Cardoso Island, São Paulo, Brazil, after accounting for phylogeny. We then evaluated specific fruit characteristics to test whether they influence consumption by birds. We found that morphological and nutritional traits showed phylogenetic signal and in general independent patterns of covariation. On the contrary, fruit color and contrast did not present phylogenetic signal and saturated fruits are rich in energy and lipids, and poor in carbohydrates. Thus we suggest that saturated fruits may indicate fruit quality. Birds consistently detected fruits with higher chromatic contrasts rather than achromatic ones. Frugivorous birds also selected lipid and energyrich fruits and fruits low defended by secondary compounds. We conclude that in spite of bird’s preferences these processes do not operate on the evolution of fruit traits, since this requires birds to differentiate between plant individual of the same and not different species. Therefore, in our study we observed that fruit traits are in general weak associate, with the exception of fruit color, that may indicate fruit adaptation to frugivores.

The response of grass, forb, and shrub seeds to the subalpine environment during the fall and under winter snow and under laboratory conditions at 20/28 C was observed for 3 consecutive years at an elevation of about 3000m on the Wasatch Plateau in central Utah. Seeds in nylon sleeves were planted under 2 cm of soil before snowfall. Under snow plantings were made directly on the soil surface and under 2 cm of soil. The seeds planted before snowfall were removed each year on four occasions: when under snow plantings were made, after snow depth exceeded 130 cm (deep snow), just before spring snowmelt, and 10 or more days following snowmelt when soils had warmed. Seeds planted under snow or under snow and soil were removed on two occasions: after snow depth exceeded 130 cm and just before the snow melted in the spring. Environmental conditions which produced seed germination varied with species, origin of species, age of seed, and temperature. Fifty-four of the 60 species planted in September under 2 cm of soil before snowfall had some seed germinated when examined just before the snow melted in the spring. Fifty of the same species planted after winter snow covered the ground in November also had some seed germination just before the snow melted in the spring. Seed germination at the near 0 C temperatures on the soil surface under snow was usually similar to the comparable seed lots also placed under 2 cm of soil. Germinability of the grasses, forbs, and shrubs studied was placed in three broad classifications: (1) Little or no seed dormancy with germination at low and warm temperatures. Seeds germinated soon after harvest at temperatures near 0 C and also in the laboratory at 20/28 C. Examples are Agropyron desertorum, A. intermedium, Chrysothamnus viscidiflorus, and Lupinus alpestris. (2) Little or no seed dormancy with germination only at warm temperatures. Seeds did not germinate at the low temperatures present in the subalpine zone during the fall and winter but germinated readily in the laboratory at 20/28 C. Examples are Aquilegia caerulea , Potentilla gracilis var. pulcherrima, Rudbeckia occidentalis, and Valeriana edulis. (3) Dormancy at seed harvest. Germination was usually increased by near 0 C temperatures, by aging, and by other environmental conditions present under deep snow. Examples are Agropyron trachycaulum, Delphinium barbeyi, Madia glomerata, and Ribes cereum var. inebrians.

Although projected terrestrial CO2 levels will not reach 1000 μmol moI-1 (0.1%) for many decades, CO2 levels in growth chambers and greenhouses routinely exceed that concentration. CO2 levels in life support systems in space can exceed 10,000 μmol moI-1 (1%) CO2. Numerous studies have examined CO2 effects up to 1000 μmol mol-1, but theoretical and some experimental evidence indicates that the beneficial effects of CO2 continue past 1000 μmol mol-1 and are near-optimal for wheat at about 1200 μmol mol-1. We studied the effects of near-optimal and superoptimal CO2 levels (>1200 μmol mol-1) on yield of two cultivars of hydroponically grown wheat in 12 trials. Increasing CO2 from suboptimal to near-optimal (350 to 1200 μmol mol-1) increased vegetative growth by 25% and seed yield by 15% in both cultivars. Yield increases were primarily the result of an increased number of heads m-2. Further elevation of CO2 to 2500 μmol mol-1reduced seed yield by 22% in cv. 'Veery-10' and by 15% in cv. 'USU-Apogee'. Superoptimal CO2 did not decrease the number of heads m-2, but reduced seeds per head by 10% and mass per seed by 11%. CO2 toxicity occurred over a wide range of light levels. Subsequent trials revealed that superoptimal CO2 in the 2 weeks before and after anthesis mimicked the effect of constant superoptimal CO2. Furthermore, near-optimal CO2 in the 2 weeks before and after anthesis mimicked the effect of constant near-optimal CO2. Nutrient concentration of leaves and heads was not affected by CO2. The yield decreases may be a response mediated by ethylene.

Biliverdin IXα is a green bile pigment produced by enzymatic cleavage of a tetrapyrrole ring of heme by heme oxygenase. While biliverdin IXα is emerging as an effective cytoprotectant, the conventional method for producing biliverdin IXα by chemical conversion of animal bile is not suitable for large scale production. A novel scalable production method was pursued via bacterial fermentation. Recombinant Escherichia coli strains were obtained by sequence optimization and plasmid transformation of a cyanobacterial heme oxygenase gene. Further strain development was done by plasmid overexpression of a native E. coli flavodoxin gene as a possible electron donor for heterogeneous heme oxygenase. The resulting strains were grown in a fed-batch culture system optimized for biliverdin IXα production. Syringomycin E is a lipodepsinonapeptide produced by certain strains of Pseudomonas syringae pv. syringae by nonribosomal peptide synthesis. Though syringomycin E had been considered a phytotoxin in the past, recent research results indicate that syringomycin E is a natural fungicide that is not toxic to animals and plants. Syringomycin E is a potential fungicide especially for use in the organic agriculture sector. New strains of P. syringae pv. syringae were isolated through ultraviolet mutagenesis and screenings for enhanced capability to produce syringomycin E especially under agitated conditions. Fermentative production was conducted in a newly formulated medium and the product was purified through a large scale chromatography system using organic-compatible solvents. Purified syringomycin E was tested on cucumber seeds to examine its antifungal activity against a soil-borne pathogen Pythium ultimum. Syringomycin E was able to inhibit Pythium infection and protected seeds and seedlings without developing disease symptoms. This dissertation research showed scalable production of two natural products, biliverdin IXα and syringomycin E in bacterial platforms. Strain development by gene recombination and mutation was done to obtain bacterial strains capable of overproducing desired metabolites. The resulting strains were grown in fermenters to maximize the yields under agitated conditions. Monitoring growth parameters and medium modifications were critical to achieve large scale production.

Fire is a recurrent disturbance that removes above ground vegetation in many locations throughout the world, including the Sydney region. Many species in fire-prone locations, and most species in the Sydney region, form soil seedbanks and regenerate through post-fire germination. However, a germination response is determined by the fire regime acting as a selective pressure over a sufficient period of time, rather than a single fire. The components of the fire-regime are intensity, season, type and frequency. The natural fire regime is dominated by warm-season fire, but management burning is conducted in cooler seasons. Cool season burning produces lower levels of germination than warm season fires in a number of locations with Mediterranean-type climate, but the effects of cool season burning on species composition in the relatively aseasonal Sydney region is unknown. An experimental approach was adopted to address this lack of knowledge. Fire can be simulated using heat shock and smoke (fire cues), and the seasonal factors of temperature and water availability can be reproduced in the laboratory. I have investigated the effect of various combinations of heat shock and smoke, of various pre-and post-fire cue temperatures, of prefire cue hydration status, of various post-fire cue water availabilities, and of accelerated aging before application of fire cues on germination of a number of species forming soil seedbanks in the Sydney region. A degree of primary dormancy was overcome in most species by the combination of heat shock and smoke in the current investigation. Fire intensity is expected to influence germination, as germination of most species was increased by the combination of heat shock and smoke within a narrow heat shock range.The interaction between ambient temperature and the level of heat shock may affect germination. Soil water content, and thus seed moisture content at the time of a fire may interact with the level of heat shock to affect both germination and survival of a seed. The age of a seed may also affect its germination response to fire. The above factors are predicted to affect the germination of species differently, and thus season of fire is expected to alter species composition. Such predictions can be readily field-tested / Doctor of Philosophy (PhD)

Fire is a recurrent disturbance that removes above ground vegetation in many locations throughout the world, including the Sydney region. Many species in fire-prone locations, and most species in the Sydney region, form soil seedbanks and regenerate through post-fire germination. However, a germination response is determined by the fire regime acting as a selective pressure over a sufficient period of time, rather than a single fire. The components of the fire-regime are intensity, season, type and frequency. The natural fire regime is dominated by warm-season fire, but management burning is conducted in cooler seasons. Cool season burning produces lower levels of germination than warm season fires in a number of locations with Mediterranean-type climate, but the effects of cool season burning on species composition in the relatively aseasonal Sydney region is unknown. An experimental approach was adopted to address this lack of knowledge. Fire can be simulated using heat shock and smoke (fire cues), and the seasonal factors of temperature and water availability can be reproduced in the laboratory. I have investigated the effect of various combinations of heat shock and smoke, of various pre-and post-fire cue temperatures, of prefire cue hydration status, of various post-fire cue water availabilities, and of accelerated aging before application of fire cues on germination of a number of species forming soil seedbanks in the Sydney region. A degree of primary dormancy was overcome in most species by the combination of heat shock and smoke in the current investigation. Fire intensity is expected to influence germination, as germination of most species was increased by the combination of heat shock and smoke within a narrow heat shock range.The interaction between ambient temperature and the level of heat shock may affect germination. Soil water content, and thus seed moisture content at the time of a fire may interact with the level of heat shock to affect both germination and survival of a seed. The age of a seed may also affect its germination response to fire. The above factors are predicted to affect the germination of species differently, and thus season of fire is expected to alter species composition. Such predictions can be readily field-tested / Doctor of Philosophy (PhD)