A aplicação de cobalto (Co) e molibdênio (Mo) diretamente às sementes de soja (Glycine max (L.) Merrill) é uma prática comum no cultivo dessa leguminosa no Brasil. Porém, esta técnica apresenta limitações, provocando fitotoxicidade na planta, interferindo na absorção de ferro (Fe) pelas raízes, diminuindo a população das bactérias fixadoras de nitrogênio do gênero Bradyrhizobium nas sementes e aumentando uma etapa no pré-plantio. Uma maneira de contornar essas limitações seria a utilização de sementes enriquecidas com Co e Mo. Contudo, há carência de pesquisas com a finalidade de aumentar o teor de Co nas sementes de soja, haja vista que o potencial de enriquecimento com Mo já foi demonstrado. O objetivo do presente projeto foi determinar a viabilidade de enriquecimento de sementes de soja com Co e Mo e relacionar o enriquecimento ao potencial fisiológico das sementes. O projeto foi composto por dois experimentos. O primeiro experimento foi desenvolvido em casa de vegetação, localizada no Centro de Energia Nuclear na Agricultura (CENA/USP), em Piracicaba (SP). O delineamento experimental utilizado foi o de blocos casualizados, com três repetições, em esquema fatorial (4x2)+1 e (4x2)+2, composto por 4 doses de Co, 2 variedades de soja, 2 formas de aplicação e o controle sem Co e Mo. Plantas de duas variedades de soja (uma de ciclo longo e outra precoce) foram cultivadas em vasos contendo 3 dm3 de amostra de solo arenoso. No estádio fenológico de desenvolvimento R5.4 (início do enchimento das sementes), aplicou-se, via solo e via folha, os seguintes tratamentos de Co + Mo (g ha-1): 0+800, 10+800, 20+800 e 30+800, além de um controle sem Co e Mo. O segundo experimento foi conduzido em campo, no Centro de Pesquisa Geraldo Schultz, em Iracemápolis (SP). O delineamento experimental foi em blocos casualizados, com 6 repetições. As doses de Co + Mo e a variedade de soja utilizadas foram idênticas às do experimento realizado em casa de vegetação, no entanto, a aplicação em campo foi exclusivamente via foliar e a variedade utilizada foi a de ciclo longo. Foram avaliados os teores de Co e Mo no solo, folhas e sementes; os índices de clorofila (IC), de flavonóides (IF) e de balanço de nitrogênio (NBI); a germinação; e o vigor das sementes enriquecidas, utilizando o método de Envelhecimento Acelerado e a análise computadorizada de plântulas, com o auxílio do software SVIS® (Seed Vigor Imaging System). Foi verificada viabilidade no enriquecimento de sementes de soja com Co e Mo, sendo que a variedade mais precoce apresentou maiores teores de Co e Mo nas sementes. O enriquecimento das sementes com Co interferiu positivamente no vigor das sementes, obtendo-se melhor desempenho com a aplicação de 20 g ha-1 de Co. O enriquecimento com Mo, no entanto, não alterou o vigor das sementes. A aplicação foliar proporcionou maiores teores de Co nas sementes, em comparação à aplicação via solo. A aplicação via solo, porém, proporcionou maiores teores de Mo nas sementes em comparação à aplicação via foliar / The application of cobalt (Co) and molybdenum (Mo) directly to soybean (Glycine max (L.) Merrill) seeds is a common practice in Brazil. However, this technique presents limitations, provoking phytotoxicity, interfering in the absorption of iron by roots, decreasing the population of nitrogen-fixing bacteria of the genus Bradyrhizobium in the seeds, and requiring an additional step in the pre-planting stage. One way to overcome these limitations would be to use seeds enriched with Co and Mo. However, there is a lack of research aimed at increasing Co content in soybean seeds, since the potential for enrichment with Mo has already been established. The aim of this study is to determine the feasibility of enriching soybean seeds with both Co and Mo and to relate the enrichment to increase in the physiological quality of the seeds. The project consists of two experiments. The first experiment was carried out under greenhouse conditions, located at the Center for Nuclear Energy in Agriculture (CENA / USP), in Piracicaba (SP). The experimental design was completely randomized, with three replications, in a factorial scheme (4x2)+1 and (4x2)+2 of which 4 Co doses, 2 varieties, 2 application modes and the control without Co and Mo. Plants of two soybean varieties (one long and one precocious cycle) were grown in pots containing 3 dm3 of sandy soil. The following treatments of Co and Mo (g ha-1) were applied: 0 + 800, 10 + 800, 20 + 800, 30 + 800, via leaf and soil, after planting, at the R5.4 phenological stage of development. The second experiment was conducted in the field, at Geraldo Schultz research center, in Iracemápolis (SP), using only the long cycle genotype. The doses of Co + Mo used were identical to those of the greenhouse experiment; however, the application was exclusively via leaf. The experimental design was in randomized blocks with 6 replications. Co and Mo content in soil, leaves and seeds as well as indices for chlorophyll (IC), flavonoids (IF), nitrogen balance (NBI), germination and vigor of enriched seeds were evaluated by means of Accelerated Aging and computer analysis of seedlings by means of SVIS® (Seed Vigor Imaging System) software. Enrichment of soybean seeds by means Co and Mo proved to be viable, with the earlier varieties having a higher Co and Mo content in the seeds. Seed vigor was positively influenced by Co enrichment; best results were obtained with application of 20 g ha-1 of Co. Enrichment with Mo, however, had no influence on seed vigor. Application via leaf resulted in higher levels of Co in seeds compared to application carried out via soil. On the other hand, application via soil resulted in higher levels of Mo in seeds in comparison to application carried out via leaf

Existem poucas informações relacionadas com a comparação de populações de soja derivadas de cruzamentos com diferentes níveis de divergência genética (DG). Os objetivos deste trabalho foram comparar o desempenho de duas populações de soja derivadas de genitores com baixa e alta DG. Foram realizados dois cruzamentos com genitores diferindo quanto à divergência genética baseada em marcadores moleculares AFLP: baixa DG (IAC-12 x IAC-100) e alta DG (EMBRAPA-60 x EMGOPA-315). Para cada cruzamento foram obtidas 100 progênies F2:3, que foram avaliadas experimentalmente em três ambientes e, posteriormente, selecionadas as 25 progênies mais produtivas. Em seguida foram obtidas 10 linhas puras F5:7 de cada uma das 25 progênies, originando aproximadamente 250 linha puras de cada cruzamento. No ano agrícola 2012/13 foram conduzidos os experimentos de avaliação, utilizando um delineamento em parcelas subdivididas, onde as progênies foram alocadas nas parcelas e as linhas puras dentro de progênies nas subparcelas, e as parcelas arranjadas em um látice balanceado 5x5 (seis repetições). As subparcelas foram constituídas de linhas de 2 m, espaçadas de 0,5 m, contendo 30 plantas após o desbaste. Os caracteres avaliados foram: número de dias para a maturação (DM), altura das plantas na maturação (AM), acamamento (AC) e produção de grãos (PG). A partir da análise de variância foram estimados os seguintes parâmetros para cada cruzamento: média geral, amplitude de variação das médias das linhas puras dentro de progênies, variância genética entre linhas puras dentro de progênies (?^2l/p ), variância fenotípica entre médias de linhas puras dentro de progênies (?^2/ F (l/p) ), coeficiente de herdabilidade entre médias de linhas puras (h ^2/X(l/p) ) e resposta esperada com seleção (Rs). Para todos os caracteres, as médias gerais foram maiores para o cruzamento de maior DG. Para PG, AM e DM as estimativas das variâncias genéticas entre linhas puras dentro de progênies F5:7 foram maiores no cruzamento de maior DG, bem como as amplitudes de variação das médias das linhas puras. Consequentemente, a resposta esperada com seleção entre médias de linhas puras dentro de progênies para PG foi 47,6% maior, em média, bem como a proporção de linhas puras de alta produção, para o cruzamento de maior DG. Os resultados deste trabalho indicam que o uso das medidas de divergência baseada em marcadores moleculares AFLP na escolha de genitores para cruzamentos pode ser uma ferramenta útil para reduzir o número de cruzamentos e, consequentemente, aumentar a eficiência da seleção para produção de grãos em soja. / There is limited information in relation to comparison of populations derived from crosses with different levels of genetic divergence (DG) in soybeans. This work was carried out to compare the performance of two soybean populations derived from parents with low and high DG. From two two-way crosses of soybeans with different levels of AFLP molecular marker genetic divergence (DG): low DG (IAC-12 x IAC-100) and high DG (EMBRAPA-60 x EMGOPA-315), one hundred F2:3 progenies were evaluated in three environments and then selected the 25 high yielding progenies. Ten inbred lines were derived in the generation F5:7 from each of 25 high yielding progeny giving rise to approximately 250 inbred lines for each cross. Evaluation trials were carried out in the 2012/13 growing season, using a split plot design, where progenies were allocated in the plots and inbred lines within progenies in the subplots, and plots arranged according to a 5x5 balanced lattice (six replications). Subplots consisted of 2 m rows spaced by 0.5 m, with 30 plants after thinning. The following traits were recorded: number of days to maturity (DM), plant height at maturity (AM), lodging (AC) and grain yield (PG). The following parameters were estimated: general mean, amplitude of variation of inbred lines within progenies means, genetic variance of inbred lines within progenies ( ?^2 l/p ), phenotypic variance on inbred lines within progenies mean basis ( ?^ 2 F(l/p) ), heritability coefficients on inbred line mean basis ( h ^2 X(l/p) ) and expected response to selection (Rs) on inbred line mean basis. For all traits, general means were higher for the high DG cross. For PG, AM and DM, genetic variance among inbred lines within F5:7 progenies were higher for the high DG cross, as well as the amplitude of variation of inbred line means. As a result, expected response to selection for grain yield (PG) was 47.6% higher, on average, as well as the proportion of high yielding inbred lines for the high DG cross. General results indicate that the use of genetically divergent parents based on AFLP molecular markers for choosing parents for crossings can be useful to reduce the number of crossings, and thus, to improve the efficiency of selection for grain yield in soybeans.

O presente trabalho tem como objetivo analisar o processo de integração produtiva da região de Dourados, localizada no sul do Estado de Mato Grosso do Sul, à economia nacional durante o período de 1976 a 2006. Uma integração que se deu pelo e para o capital, com a inserção dessa região ao novo padrão produtivo nacional que direcionou os produtores para as modernas lavouras de grãos, principalmente de soja. Essa lavoura não foi a única atividade produtiva que conferiu à região uma integração econômica, seja ao mercado nacional seja ao internacional. Desde o final da Guerra do Paraguai (1870) até meados dos anos 70, houve, pelo menos, duas outras formas de integração econômica regional. Uma delas se processou com as tradicionais atividades da pecuária bovina e do extrativismo da erva-mate ocorridas no período de 1870 a 1937, que configurou o território regional em grandes propriedades rurais voltadas ao mercado externo; a outra se processou com a necessidade da formação de um mercado de produção agrícola e de consumo de produtos industriais, que reconfigurou o território regional em pequenas propriedades rurais frutos dos projetos de colonização ocorridos no período de 1943 a 1956. A partir de 1976, com a criação do Programa Especial de Desenvolvimento da Região da Grande Dourados, as relações capitalistas de produção que se consolidaram na região deixaram de serem pautadas quase que exclusivamente pelas tradicionais atividades da pecuária bovina e do extrativismo da erva mate, para se constituírem num novo eixo de acumulação vinculado às modernas lavouras de grãos. Ao constituir esse novo eixo de acumulação, a região de Dourados foi conduzida a um processo de integração seletivo e excludente do ponto de vista sócio-territorial. Nesse processo, parte dos produtores mudaram seu eixo de acumulação para as modernas lavouras de grãos sendo inseridos à nova dinâmica produtiva nacional integrada ao novo padrão agrário de produzir. / This study aims to analyze the productive integration process of Dourados region, located in the southern of Mato Grosso do Sul, in the nacional economy during the period 1976 to 2006. Integration that has by and for the working capital with the insertion of this new pattern of the national production, which directed the producers to modern grain crops, especially soybeans. Since the end of the Paraguayan War (1870) until the mid 70s of last century, there was, at least, two other economic integration forms, one of them took place with the traditional activities of cattle raising and extraction of mate occurred on the period 1870 to 1937, who set up the regional territory in large farms aimed at foreign markets, the other was processed with the formation need of a market for agricultural production and consumption of industrial products, which reconfigured the regional territory in small fruit farms in the settlement projects occurred during the period 1943 to 1956. Since 1976, with the creation of Prodegran (Programa Especial de Desenvolvimento da Região da Grande Dourados - Special Program for the Great Dourados Development), capitalist relations of production that have been consolidated in the region was no longer guided almost exclusively by traditional activities of cattle and the extraction of the herb mate, to constitute a new axis of accumulation linked to the modern grain crops. As an axis of accumulation, the Dourados region has led to an integration process from the point of socio-territorial, selective and exclusive, that part of the producers changed their accumulation axis for modern grain crops integrated to the new pattern of national production.

Guan, Lei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 123-145). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABSTRACT --- p.II / LIST OF ABBREVIATIONS --- p.VII / TABLE OF CONTENT --- p.IIX / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Distribution and Origins --- p.1 / Chapter 1.3 --- History of Use --- p.2 / Chapter 1.4 --- Chemical Structure --- p.3 / Chapter 1.5 --- Physiologic Properties --- p.5 / Chapter 1.6 --- Absorption and Metabolism --- p.7 / Chapter 1.6.1 --- Concentration of Isoflavones in Plasma --- p.10 / Chapter 1.6.2 --- Urinary Excretion --- p.10 / Chapter 1.7 --- Healthy Effects --- p.11 / Chapter 1.7.1 --- Menopausal Symptom --- p.11 / Chapter 1.7.2 --- Cardiovascular Disease --- p.12 / Chapter 1.7.3 --- Osteoporosis --- p.13 / Chapter 1.7.4 --- Tumors --- p.14 / Chapter 1.7.4.1 --- Breast Cancer --- p.14 / Chapter 1.7.4.2 --- Prostate Cancer --- p.15 / Chapter 1.7.5 --- Alcohol Addiction --- p.16 / Chapter 1.7.6 --- Potential Adverse Effects --- p.16 / Chapter 1.8 --- Summary --- p.17 / Chapter Chapter 2 --- Determination of Isoflavones in Soybean and Gegen --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.1.1 --- Classification and Structure of Phytoestrogens --- p.19 / Chapter 2.1.2 --- Isoflavones in Soybeans and Gegen --- p.21 / Chapter 2.1.3 --- Methods of Determination --- p.26 / Chapter 2.1.3.1 --- Isolation and Purification of Isoflavones --- p.26 / Chapter 2.1.3.2 --- Analytical Methods for Isoflavones in Soybeans and Gegen --- p.26 / Chapter 2.2 --- Objective --- p.28 / Chapter 2.3 --- Materials and Methods --- p.29 / Chapter 2.3.1 --- Extraction and Isolation of Soybean and Gegen Isoflavone Extracts --- p.29 / Chapter 2.3.2 --- HPLC Analysis --- p.29 / Chapter 2.3.2.1 --- Sample Preparation for the HPLC Analysis --- p.29 / Chapter 2.3.2.2 --- HPLC Analysis --- p.30 / Chapter 2.3.3.3 --- Qualitative Analysis of the Isoflavones and Their Glycosides in Soybean and Gegen --- p.30 / Chapter 2.4 --- Results --- p.31 / Chapter 2.4.1 --- Isoflavone Identification of Soybean Extract --- p.31 / Chapter 2.4.2 --- Isoflavone Identification of Gegen Extract --- p.33 / Chapter 2.5 --- Discussion --- p.35 / Chapter Chapter 3 --- Hypocholesterolemic Effects of Soybean and Gegen Isoflavone Extracts in Ovariectomized，Intact Male and Castrated Golden Syrian Hamsters --- p.38 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Objective --- p.41 / Chapter 3.3 --- Materials and Methods --- p.42 / Chapter 3.3.1 --- Preparation of Soybean and Gegen Isoflavone Extracts --- p.42 / Chapter 3.3.2 --- Animals and Diets --- p.42 / Chapter 3.3.3 --- Serum Lipid and Lipoprotein Determinations --- p.45 / Chapter 3.3.4 --- Determination of Cholesterol Concentration in the Organs --- p.45 / Chapter 3.3.5 --- Statistics --- p.46 / Chapter 3.4 --- Results --- p.48 / Chapter 3.4.1 --- Food Intake and Body and Relative Organ Weights --- p.48 / Chapter 3.4.2 --- Effects of Soybean and Gegen Isoflavone Extracts on Serum and Organ Cholesterol in Ovariectomized and Intact Male and Castrated Hamsters --- p.56 / Chapter 3.5 --- Discussion --- p.61 / Chapter Chapter 4 --- Possible Developmental and Reproductive Toxicity of Soybean Isoflavones on SD Rats --- p.67 / Chapter 4.1 --- Introduction --- p.67 / Chapter 4.2 --- Objective --- p.70 / Chapter 4.3 --- Materials and Methods --- p.71 / Chapter 4.3.1 --- Diet --- p.71 / Chapter 4.3.2 --- Animals --- p.73 / Chapter 4.3.3 --- Study Design --- p.73 / Chapter 4.3.4 --- Measurement of Reproductive Hormones --- p.74 / Chapter 4.3.5 --- Measurement of Sperm Number --- p.74 / Chapter 4.3.6 --- Statistics --- p.75 / Chapter 4.4 --- Results --- p.77 / Chapter 4.4.1. --- Food Intake and Food Efficiency Ratio --- p.77 / Chapter 4.4.2 --- Growth Trend --- p.79 / Chapter 4.4.3 --- Organ Weight --- p.82 / Chapter 4.4.3.1 --- Absolute Organ Weight --- p.82 / Chapter 4.4.3.2 --- Relative Organ Weight --- p.84 / Chapter 4.4.4 --- Reproductive Hormone Levels --- p.86 / Chapter 4.4.5 --- Epididymis Parameters of Male Rats --- p.88 / Chapter 4.5 --- Discussion --- p.90 / Chapter Chapter 5 --- Possible Developmental and Reproductive Toxicity of Gegen Isoflavones on SD Rats --- p.97 / Chapter 5.1 --- Introduction --- p.97 / Chapter 5.2 --- Objective --- p.99 / Chapter 5.3 --- Materials and Methods --- p.100 / Chapter 5.3.1 --- Animals and Diets --- p.100 / Chapter 5.3.2 --- Study Design --- p.100 / Chapter 5.3.3 --- Statistics --- p.101 / Chapter 5.4 --- Results --- p.103 / Chapter 5.4.1 --- Food Consumption and Food Efficiency Ratio --- p.103 / Chapter 5.4.2 --- Growth Trend --- p.105 / Chapter 5.4.3 --- Organ Weights --- p.108 / Chapter 5.4.3.1 --- Absolute Organ Weights --- p.108 / Chapter 5.4.3.2 --- Relative Organ Weight --- p.110 / Chapter 5.4.4 --- Reproductive Hormone Levels --- p.112 / Chapter 5.4.5 --- Epididymis Parameters of Male Rats --- p.114 / Chapter 5.5 --- Discussion --- p.116 / Chapter Chapter 6 --- Conclusions --- p.121 / References --- p.123

Submitted by admin tede (tede@pucgoias.edu.br) on 2018-08-23T14:48:51Z No. of bitstreams: 1 Patrícia Regina da Silva Zaluski.pdf: 1284798 bytes, checksum: 2ae114d5a9bdb1ae65916591abc56a0d (MD5) / Made available in DSpace on 2018-08-23T14:48:51Z (GMT). No. of bitstreams: 1 Patrícia Regina da Silva Zaluski.pdf: 1284798 bytes, checksum: 2ae114d5a9bdb1ae65916591abc56a0d (MD5) Previous issue date: 2018-02-08 / The traditional logistics seeks reduction of costs with greater efficiency in the service, attending to the dimensions of performance speed, flexibility and cost. When the sustainability theme is inserted into logistics management, there are emerging analyzes of atmospheric CO2 emissions and their cost impacts on transportation modes. In this context, the main objective of this research was to analyze the efficiency of the modal arrangements of the outflow of soybeans produced in Brazil for export, and CO2 emissions for an intermodal transport network of soybeans exported in Brazil. The emission estimate was calculated based on the adapted formula of ANTT (2014a; 2014b) reports, which followed methodologies presented by the IPCC and MCTI (2010). The method consists in the estimation of emissions by consumption factor of diesel oil, considering the tonnage transported in TKU. The results obtained show the best export routes for soybeans, considering freight costs and CO2 emissions. The Brazilian logistics infrastructure needs investments in the construction of new terminals, in order to favor the use of intermodality. The road mode still has lower logistics costs and holds greater supply and availability in the market, in contrast, intermodality has the best performance in reducing emissions. / A logística tradicional busca redução de custos com maior eficiência no serviço, atendendo às dimensões de desempenho velocidade, flexibilidade e custo. Quando o tema sustentabilidade é inserido à gestão logística, emergem análises sobre as emissões atmosféricas de CO2 e seus impactos em termos de custos nos modais de transporte. Nesse contexto, o objetivo central desta pesquisa foi analisar a eficiência dos arranjos modais de escoamento da soja produzida no Brasil destinada à exportação, e de emissões de CO2 para uma rede de transporte intermodal da soja de exportação no Brasil. A estimativa de emissões foi calculada com base na fórmula adaptada de relatórios da ANTT (2014a; 2014b), que seguiu metodologias apresentadas pelo IPCC e MCTI (2010). O método consiste na estimação de emissões por fator de consumo de óleo diesel, considerando a tonelagem transportada em TKU. Os resultados obtidos apresentam as melhores rotas de escoamento da soja de exportação, considerando os custos de frete e emissões de CO2. A infraestrutura logística brasileira carece de investimentos na construção de novos terminais, de modo a favorecer o uso da intermodalidade. O modo rodoviário ainda possui menores custos logísticos e detém maior oferta e disponibilidade no mercado, em contrapartida, a intermodalidade possui os melhores desempenhos em redução de emissões.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 117-126).

Bean leaf beetle, Cerotoma trifurcata (Forster) (Coleoptera: Chrysomelidae), is an economic pest of soybean in Ontario. Field cage studies were conducted in 2010-2011 to determine voltinism in southern Ontario and the effect of C. trifurcata feeding during soybean reproductive stages on soybean yield and quality. Thermal requirements for C. trifurcata development were determined in a laboratory study. Results of field and laboratory studies support the occurrence of one generation of C. trifurcata in southern Ontario. Pod-feeding increased with both number of beetles and soybean reproductive stage. An increase in defoliation and a reduction in seed quality were observed with increasing number of beetles. Monitoring programs for late-season pod-feeding should begin when degree days approach 500 (base 9.31°C). Economic thresholds for C. trifurcata during soybean reproductive stages range from 0.27 to 2.00 beetles per plant or 8 to 60 beetles per m of row. / Ontario Ministry of Agriculture and Food – University of Guelph Sustainable Production (Plants) Program and Grain Farmers of Ontario through the Farm Innovation Program.

Legume-rhizobia symbioses contribute at least 20% of the biosphere’s supply of reactive nitrogen. These unique associations rely on the exchange of specific molecular signals between nitrogen-fixing soil bacteria, collectively called rhizobia, and their host plants and, with few exceptions, result in the formation of root nodules, which provide an environment suitable for nitrogen fixation. However, nitrogen fixation is energetically expensive and nodule proliferation, in much the same manner as the proliferation of other meristems in plants, must be controlled in order to attain equilibrium between cell proliferation and differentiation. Nodule proliferation is controlled primarily and systemically by autoregulation of nodulation (AON). A gene central to this process was first isolated by map-based cloning from soybean (Glycine max) and was named G. max Nodule Autoregulation Receptor Kinase (GmNARK) in accordance with its biochemical and physiological functions. Expression patterns of GmNARK have been described by several investigators; however, these reports were based on either non-quantitative methods or a limited number of tissue types. More importantly, the expression domains of GmNARK were completely unknown. The study described in this thesis utilised techniques such as quantitative RT-PCR (QRTPCR), transcription start site mapping, promoter-reporter gene fusion, and promoter deletion, to analyse the expression levels and domains of GmNARK across a variety of tissues as well as identify the promoter elements that are responsible for the basal and tissue-specific expression of GmNARK. In addition, the promoter activity of GmNARK was also compared with that of Lotus japonicus HAR1, the GmNARK orthologue, in both homologous and heterologous transformation systems. Based on QRT-PCR, GmNARK was expressed to varying levels throughout the plant; the transcript was detected at high levels in mature leaves and roots but to a lesser extent in young leaves, shoot tips and nodules. The transcript level was not significantly affected by Bradyrhizobium japonicum during the first week following inoculation. Histochemical analysis of L. japonicus plants carrying either a 1.7 kb GmNARK promoter or 2.0 kb LjHAR1 promoter fused to a -glucuronidase reporter gene localised GUS activity to living cells within vascular bundles, especially phloem cells in leaves, stems, roots, and nodules. Phloem-specific expression was also detected in soybean hairy roots carrying these constructs. These results suggested that both cis- and trans-acting elements required for the transcriptional regulation of these orthologous genes are likely to be conserved. In contrast, 1.7 kb of the GmNARK promoter did not drive phloem-specific expression in Arabidopsis thaliana, indicating the absence of the trans-acting elements required for the tissue-specificity of GmNARK in this distantly related species. The comparison of 2.0 kb of promoter sequences of GmNARK, LjHAR1 and Medicago truncatula SUNN, another GmNARK orthologue, using bioinformatics and computational approaches indicated several highly conserved motifs including a putative negative regulatory region (NRR), which was previously reported to repress gene expression in non-phloem cell types. Deletion analysis of the GmNARK promoter, however, ruled out the possibility that this motif, found at -308 bp with respect to the translation start site, was truly functional and located the region controlling phloem-specific expression to DNA sequence between 908 bp and 1.7 kb upstream of the start codon. Two other candidate regions were identified by Multiple EM for Motif Elicitation (MEME). These regions, namely MEME3 and MEME4 showed strong sequence similarity to the corresponding regions of the LjHAR1 promoter. Interestingly, the MEME3 motif was also found in the MtSUNN promoter at a similar location to that of LjHAR1. Potential NRRs in the LjHAR1 and MtSUNN promoters were found in the MEME3 motifs, whereas only a variant form of a NRR in the GmNARK promoter was found in this region. Additionally, an identical semi-palindromic sequence was also observed in the MEME3 motifs of the three orthologous promoters. Based on these findings, the semi-palindromic sequence and the variant form of the NRR are proposed to be positive and negative regulatory elements for the phloem-specific expression of GmNARK, respectively. The computational approaches also identified two potential TATA elements in the GmNARK promoter. Rapid amplification of 5’ cDNA ends and promoter deletion analysis have confirmed that they were functional. The two TATA elements in GmNARK promoter appeared to cooperatively direct transcription of GmNARK, but either was adequate for basal transcription. The finding that the expression of AON receptor-like kinase genes is phloem-specific has contributed to a better understanding of AON signalling pathways. The QRT-PCR study and the discovery of cis-acting regulatory regions have also provided crucial information on the transcriptional regulation of GmNARK as well as plant genes in general. Additionally, the promoters of GmNARK and LjHAR1 could potentially be used to drive phloem-specific expression in legume biotechnology research.

[Truncated abstract] Nitrogen fixation is a feature of the symbiotic association between legumes and rhizobia, which occurs within the symbiosomes of root nodules and involves the conversion of atmospheric N2 to ammonia to be used by the plant in exchange for carbon compounds. Exchange of other nutrients is controlled by plant-synthesised proteins on the symbiosome membrane. Iron is a component of symbiotically important proteins, so is essential for nitrogen fixation. Low soil iron leads to decreased plant yields, whilst in other environments plants may accumulate iron to toxic levels. Knowledge of iron acquisition, transport and storage mechanisms is important to elucidate the role of iron transporters in the maintenance of iron homeostasis in the plant. This study provides evidence that iron has a profound effect in the Bradyrhizobium japonicum-Glycine max symbiosis on the development of the nodule, and on the development of the symbiotic soybean plant itself. cDNAs encoding four putative iron transporters in soybean; GmDmt1, GmYSL1, GmCCC1;1 and GmCCC1;2, were identified, isolated and characterised in this study. GmDmt1 is localised to the symbiosome membrane. Expression of GmDmt1 occurs in nodules, roots and leaves and increases in response to iron starvation. GmDmt1 rescues growth and enhances 55Fe(II) uptake in the iron transport deficient yeast strain fet3fet4, with uptake following Michaelis-Menten kinetics, resembling the situation in isolated symbiosomes. Competition experiments using fet3fet4 indicated that GmDmt1 is able to transport other divalent cations, including zinc, copper and manganese, and is also able to complement a zinc transport deficient yeast mutant. ... These results suggest the divalent metal transporter GmDmt1, the putative iron chelate transporter GmYSL1 and the putative vacuolar iron transporters GmCCC1;1 and GmCCC1;2 act together to maintain iron homeostasis in symbiotic soybeans. The possible interactions and regulation of these proteins and their roles in the acquisition, transport and utilisation of iron in symbiotic soybeans are discussed.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.