Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The ingestive behavior and the ingestion of forage by beef heifers were evaluated in the vegetative and flowering stages of ryegrass (Lolium multiflorum L.). The treatments were: grazing exclusively on ryegrass, ryegrass intercropping with vetch (Vicia sativa L.) or red clover (Trifolium pratense L.). The grazing method was continuous with variable stocking. The experimental design was the completely randomized with repeated measures arrangement, three forage systems and three repetitions of area. For evaluations of forage intake, chromic oxide was used as an indicator of fecal production and heifers were considered as experimental units. The forage mass, allowance of forage and allowance of leaf blades were, 1596.2 kg / ha DM, 12 kg DM / 100 kg BW and 4.5 kg DM / 100 kg BW respectively. The participation of vetch and red clover in the forage mass corresponded to 13.2% and 4.5% respectively in the vegetative stage. In the flowering stage, the participation of legumes corresponded to 0.6%. In forage as grazed, crude protein (17.8%), neutral detergent fiber (55.8%) and in situ dry matter digestibility (76.7%) were similar among forage systems. These variables differed between phenological stages of ryegrass. The grazing time differed between the systems being superior in the system with vetch. The forage intake rate in the clover system was higher than the other systems. The dry matter intake was similar among forage systems and was higher in the vegetative stage compared to the flowering stage. The average daily gain was similar among forage systems and phenological stages. With little participation of red clover or vetch in the herbage mass intercropped with ryegrass, it does not modify the intake of dry matter, crude protein and neutral detergent fiber, resulting in the same performance of beef heifers. / Foram avaliados o comportamento ingestivo e a ingestão de forragem por bezerras de corte, nos estádios vegetativo e de florescimento do azevém (Lolium multiflorum L.). Os tratamentos foram: pastagem exclusiva de azevém, azevém consorciado com ervilhaca (Vícia sativa L.) ou com trevo vermelho (Trifolium pratense L.). O método de pastejo foi o contínuo com lotação variável. O delineamento experimental foi o inteiramente casualizado com medidas repetidas no tempo, três sistemas forrageiros e três repetições de área. Para as avaliações da estimativa de ingestão de forragem, foi utilizado o óxido de cromo como indicador da produção fecal e as bezerras foram consideradas as unidades experimentais. A massa de forragem, oferta de forragem e oferta de lâminas foliares foram: 1596,2 kg/ha de MS, 12 kg de MS/100 kg PC e 4,5 kg de MS/ 100 kg PC, respectivamente. A participação de “ervilhaca” e “trevo vermelho” na massa de forragem correspondeu a 13,2% e 4,5% respectivamente no estádio vegetativo. No estádio de florescimento o percentual das leguminosas correspondeu a 0,6%. Na forragem da simulação do pastejo, a proteína bruta (17,8%), fibra em detergente neutro (55,8%) e digestibilidade in situ da matéria seca (76,7%), foram similares entre os sistemas forrageiros. Essas variáveis diferiram entre estádios fenológicos do azevém. O tempo de pastejo diferiu entre os sistemas sendo superior no sistema com ervilhaca. A taxa de ingestão de forragem no sistema com trevo foi superior aos demais sistemas. A ingestão de matéria seca foi similar entre os sistemas forrageiros e foi maior no estádio vegetativo em relação ao estádio de florescimento. O ganho médio diário foi semelhante entre sistemas forrageiros e estádios fenológicos. Com pequena participação de “trevo vermelho” ou “ervilhaca” na massa de forragem consorciada com azevém, a ingestão de matéria seca, de proteína bruta e de fibra em detergente neutro não são modificadas resultando em mesmo desempenho de bezerras de corte.

O trevo-vermelho (Trifolium pratense L.), uma das leguminosas forrageiras mais utilizadas na agricultura mundial, contém as isoflavonas formononetina e biochanina A, e em menores concentrações daidzeína e genisteína. Estes compostos têm ganhado muito interesse devido aos relatos de seus benefícios à saúde humana. Recentemente, no ano de 2009, esta espécie foi incluida na lista da Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) que contém plantas medicinais com potencial de gerar produtos para serem produzidos e distribuídos pelo SUS. Mas é importante destacar que não existem estudos de quantificação de isoflavonas em plantas de trevo-vermelho cultivados no Brasil. Os objetivos deste trabalho foram validar método de análise de amostras de trevo-vermelho por Cromatografia Líquida de Alta Eficiência (CLAE); quantificar quatro isoflavonas em 78 acessos de trevo-vermelho; verificar as modificações sazonais na concentração de três acessos de trevo-vermelho; aumentar os níveis de isoflavonas através de melhoramento genético; e determinar a atividade anti-inflamatória in vivo e in vitro e de atividade de inibição da acetilcolinesterase de um extrato seco de trevo-vermelho. Para analisar as plantas por CLAE, estas foram hidrolisadas, separadas por diclorometano, e ressuspendidas em metanol. O método de CLAE usado foi validado. As isoflavonas (daidzeína, genisteína, formononetina e biochanina A) foram quantificadas em 78 acessos de trevo-vermelho, os conteúdos das agliconas (expressos em μg/g de planta seca, n=3) destes compostos variaram entre 0,00 a 137,91 para daidzeína; 14,70 a 516,51 para genisteína; 452,97 a 28548,65 para formononetina; e 1967,64 a 20145,27 para biochanina A, e a concentração total de isoflavonas variou entre 9,81 e 36,36 mg/g. O estudo sazonal mostrou que a concentração total das isoflavonas não difere entre as estações, mas verifica-se que na primavera (quando o trevo-vermelho está no estágio reprodutivo) se observam concentrações inferiores, e no inverno (quando a planta está no estágio vegetativo) são encontradas concentrações mais elevadas. No estudo de melhoramento genético foi possível observar um aumento na concentração total de isoflavonas. A atividade anti-inflamatória in vitro foi medida através da mobilização de leucócitos, pelo ensaio de quimiotaxia na câmara de Boyden. A atividade anti-inflamatória in vivo foi avaliada pelo teste de edema de pata de rato induzido por carragenina. Os resultados do teste anti-inflamatório in vitro mostram que houve significante inibição da migração dos leucócitos nas concentrações de 100,0 (94,73% de inibição), 50,0 (95,39% de inibição), 25,0 (94,73% de inibição), 10,0 (84,68% de inibição) e 5,0 (78,75% de inibição) μg/mL de extrato seco de trevo-vermelho. O teste anti-inflamatório in vivo demonstrou significante atividade nas doses testadas, 100 e 50 mg/kg de extrato seco de trevo-vermelho. O percentual médio de inibição do edema foi 63,37%. O teste da inibição da acetilcolinesterase não demonstrou atividade. Os resultados deste estudo sugerem que se pode produzir plantas com concentrações mais elevadas de isoflavonas para a produção de fitomedicamentos com maior qualidade, e o extrato de trevo-vermelho pode ser adequado para o tratamento de doenças inflamatórias. / Red clover (Trifolium pratense L.) is one of the most utilized forage legume in the world agriculture and contains the isoflavones formononetin and biochanin A, and in smaller concentration daidzein and genistein. These compounds have gained a high interest due to their human health benefits. Recently this species was included in the list of Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) which contains medicinal plants that have potential to generate products to be produced and distributed by SUS. But it is important to highlight that does not exist any study of isoflavones quantification in red clover plants cultivated in Brazil. The aims of this work were to validate a method to analize red clover samples by High Performance Liquid Cromatography (HPLC); to quantify four isoflavones compounds in 78 red clover accessions; to verify seasonal modifications in the isoflavone concentration of three red clover accessions; to increase isoflavones levels through plant breeding; and to determine in vivo and in vitro anti-inflammatory activity, and inhibitory activity of acetylcholinesterase of a red clover dry extract. To analyze the plants by HPLC, they were hydrolyzed, separated by dichloromethane, and resuspended with methanol. The HPLC method used was validated. Isoflavones (daidzein, genistein, formononetin and biochanin A) were quantified in 78 red clover accessions, the aglycone contents (expressed in μg/g of dry material, n=3) of these compounds varied from 0.00 to 137.91 to daidzein; 14.70 to 516.51 to genistein; 452.97 to 28548.65 to formononetin; and 1967.64 to 20145.27 to biochanin A, and the total isoflavone concentration ranged between 9.81 and 36.36 mg/g. The seasonal study showed that the total concentration of isoflavones is not statistically different at different seasons, but is possible to verify that at spring (when red clover is at reproductive stage) we could observe the lowest concentration, and at winter (when red clover is at vegetative stage) we found the highest concentration. In the breeding study we could observe an increase in the total concentration of isoflavones. The in vitro anti-inflammatory activity was measured towards by leucocytes mobilization, by chemotaxis assay in Boyden´s chamber. The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at concentrations of 100.0 (94.73% of inhibition), 50.0 (95.39% of inhibition), 25.0 (94.73% of inhibition), 10.0 (84.68% of inhibition) and 5.0 (78.75% of inhibition) μg/mL of red clover dry extract. The in vivo anti-inflammatory test demonstrated significant activity in the tested doses, 100 and 50 mg/kg of red clover dry extract. The average of edema inhibition percentage was 63.37%. The inhibition of acetylcholinesterase test did not show activity. The results of this study suggest that we can select or produce plants with higher concentrations of isoflavones to produce phytomedicines with higher quality, and the red clover extract might be suitable for the treatment of inflammatory diseases.

O trevo-vermelho (Trifolium pratense L.), uma das leguminosas forrageiras mais utilizadas na agricultura mundial, contém as isoflavonas formononetina e biochanina A, e em menores concentrações daidzeína e genisteína. Estes compostos têm ganhado muito interesse devido aos relatos de seus benefícios à saúde humana. Recentemente, no ano de 2009, esta espécie foi incluida na lista da Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) que contém plantas medicinais com potencial de gerar produtos para serem produzidos e distribuídos pelo SUS. Mas é importante destacar que não existem estudos de quantificação de isoflavonas em plantas de trevo-vermelho cultivados no Brasil. Os objetivos deste trabalho foram validar método de análise de amostras de trevo-vermelho por Cromatografia Líquida de Alta Eficiência (CLAE); quantificar quatro isoflavonas em 78 acessos de trevo-vermelho; verificar as modificações sazonais na concentração de três acessos de trevo-vermelho; aumentar os níveis de isoflavonas através de melhoramento genético; e determinar a atividade anti-inflamatória in vivo e in vitro e de atividade de inibição da acetilcolinesterase de um extrato seco de trevo-vermelho. Para analisar as plantas por CLAE, estas foram hidrolisadas, separadas por diclorometano, e ressuspendidas em metanol. O método de CLAE usado foi validado. As isoflavonas (daidzeína, genisteína, formononetina e biochanina A) foram quantificadas em 78 acessos de trevo-vermelho, os conteúdos das agliconas (expressos em μg/g de planta seca, n=3) destes compostos variaram entre 0,00 a 137,91 para daidzeína; 14,70 a 516,51 para genisteína; 452,97 a 28548,65 para formononetina; e 1967,64 a 20145,27 para biochanina A, e a concentração total de isoflavonas variou entre 9,81 e 36,36 mg/g. O estudo sazonal mostrou que a concentração total das isoflavonas não difere entre as estações, mas verifica-se que na primavera (quando o trevo-vermelho está no estágio reprodutivo) se observam concentrações inferiores, e no inverno (quando a planta está no estágio vegetativo) são encontradas concentrações mais elevadas. No estudo de melhoramento genético foi possível observar um aumento na concentração total de isoflavonas. A atividade anti-inflamatória in vitro foi medida através da mobilização de leucócitos, pelo ensaio de quimiotaxia na câmara de Boyden. A atividade anti-inflamatória in vivo foi avaliada pelo teste de edema de pata de rato induzido por carragenina. Os resultados do teste anti-inflamatório in vitro mostram que houve significante inibição da migração dos leucócitos nas concentrações de 100,0 (94,73% de inibição), 50,0 (95,39% de inibição), 25,0 (94,73% de inibição), 10,0 (84,68% de inibição) e 5,0 (78,75% de inibição) μg/mL de extrato seco de trevo-vermelho. O teste anti-inflamatório in vivo demonstrou significante atividade nas doses testadas, 100 e 50 mg/kg de extrato seco de trevo-vermelho. O percentual médio de inibição do edema foi 63,37%. O teste da inibição da acetilcolinesterase não demonstrou atividade. Os resultados deste estudo sugerem que se pode produzir plantas com concentrações mais elevadas de isoflavonas para a produção de fitomedicamentos com maior qualidade, e o extrato de trevo-vermelho pode ser adequado para o tratamento de doenças inflamatórias. / Red clover (Trifolium pratense L.) is one of the most utilized forage legume in the world agriculture and contains the isoflavones formononetin and biochanin A, and in smaller concentration daidzein and genistein. These compounds have gained a high interest due to their human health benefits. Recently this species was included in the list of Relação Nacional de Plantas Medicinais de Interesse ao SUS (RENISUS) which contains medicinal plants that have potential to generate products to be produced and distributed by SUS. But it is important to highlight that does not exist any study of isoflavones quantification in red clover plants cultivated in Brazil. The aims of this work were to validate a method to analize red clover samples by High Performance Liquid Cromatography (HPLC); to quantify four isoflavones compounds in 78 red clover accessions; to verify seasonal modifications in the isoflavone concentration of three red clover accessions; to increase isoflavones levels through plant breeding; and to determine in vivo and in vitro anti-inflammatory activity, and inhibitory activity of acetylcholinesterase of a red clover dry extract. To analyze the plants by HPLC, they were hydrolyzed, separated by dichloromethane, and resuspended with methanol. The HPLC method used was validated. Isoflavones (daidzein, genistein, formononetin and biochanin A) were quantified in 78 red clover accessions, the aglycone contents (expressed in μg/g of dry material, n=3) of these compounds varied from 0.00 to 137.91 to daidzein; 14.70 to 516.51 to genistein; 452.97 to 28548.65 to formononetin; and 1967.64 to 20145.27 to biochanin A, and the total isoflavone concentration ranged between 9.81 and 36.36 mg/g. The seasonal study showed that the total concentration of isoflavones is not statistically different at different seasons, but is possible to verify that at spring (when red clover is at reproductive stage) we could observe the lowest concentration, and at winter (when red clover is at vegetative stage) we found the highest concentration. In the breeding study we could observe an increase in the total concentration of isoflavones. The in vitro anti-inflammatory activity was measured towards by leucocytes mobilization, by chemotaxis assay in Boyden´s chamber. The in vivo anti-inflammatory activity was tested by a carrageenan-induced rat paw edema test. The results of anti-inflammatory in vitro test showed that there was a significant inhibition of leukocyte migration at concentrations of 100.0 (94.73% of inhibition), 50.0 (95.39% of inhibition), 25.0 (94.73% of inhibition), 10.0 (84.68% of inhibition) and 5.0 (78.75% of inhibition) μg/mL of red clover dry extract. The in vivo anti-inflammatory test demonstrated significant activity in the tested doses, 100 and 50 mg/kg of red clover dry extract. The average of edema inhibition percentage was 63.37%. The inhibition of acetylcholinesterase test did not show activity. The results of this study suggest that we can select or produce plants with higher concentrations of isoflavones to produce phytomedicines with higher quality, and the red clover extract might be suitable for the treatment of inflammatory diseases.

The objective of this research was to evaluate three grazing systems with Coastcross-1 (CC) + 100 kg N/ha/year + common vetch; CC + 100 kg N/ha/year + arrowleaf clover; and CC + 200 kg N/ha/year. The experiment was carried out from May 2013 to April 2014. Lactating Holstein cows were used in the evaluation. The grazing method was the rotative stocking, with one day of occupation. Thirteen grazing cycles were performed during the experimental period (313 days). The forage mass, botanical composition, leaf/stem ratio; daily accumulation rate, production and forage intake; grazing efficiency and stocking rate were evaluated. Experimental design was completely randomized with three treatments (grazing systems), three replicates (paddocks) in completely split-plot time (seasons). The average of forage production and stocking rate were 20.8, 17.6 and 19.7 t/ha/year; 7.0, 6.8 e 6.8 animal units/ha/day, respectively. Better results were found on CC + 100 kg of N/ha + common vech and CC + 200 kg of N/ha pasture systems. The presence of the legumes did not affect the structural composition of Coastcross-1, but implies delay in the development of this grass. / Objetivou-se com esta pesquisa avaliar três sistemas forrageiros constituídos por: Coastcross-1(CC) + ervilhaca comum + 100 kg de N/ha/ano; CC + trevo vesiculoso + 100 kg de N/ha/ano e CC + 200 kg de N/ha/ano. O experimento foi conduzido entre maio de 2013 e abril de 2014. Para avaliação foram utilizadas vacas em lactação da raça Holandesa. O método de pastejo utilizado foi o de lotação rotacionada, com um dia de ocupação. Durante o período experimental (313 dias) foram realizados treze pastejos. Foram avaliados a massa de forragem, composição botânica, relação folha/colmo; taxa de acúmulo diário, produção e consumo de foragem; eficiência de pastejo e taxa de lotação. O delineamento experimental utilizado foi o inteiramente casualizado com três tratamentos (sistemas forrageiros), três repetições (piquetes) e parcelas subdivididas no tempo (estações do ano). Os valores médios de produção de forragem e taxa de lotação foram de 20,8; 17,6 e 19,7 t/ha/ano; 7,0; 6,8 e 6,8 unidades animais/ha/dia, respectivamente. Melhores resultados foram encontrados nos sistemas forrageiros constituídos por CC + 100 kg de N/ha + ervilhaca comum e CC + 200 kg de N/ha. A presença das leguminosas não afetou a composição estrutural da Coastcross-1, mas implica em atraso no desenvolvimento dessa gramínea.

Phosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.

Phosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.

The investigation in this thesis is divided into two parts. In the first part, the expression and accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO), the enzyme which catalyses the final step of ethylene biosynthesis in higher plants, is examined during exposure of white clover (Trifolium repens L.) to a water deficit. The second part of this thesis is focused on the identification and characterisation of a water-deficit-associated ACC synthase (ACS), the enzyme which catalyses the production of ACC. In the first part, two white clover varieties with differing sensitivity to water deficit, a drought-tolerant Tienshan ecotype and a drought-sensitive Grasslands Challenge cv. Kopu II cultivar were exposed to two water deficit treatments: one cycle of water deficit (designated non-prestressed; NPS) and a water deficit, a rehydration period and then a second water deficit treatment (designated pre-stressed; PS) in the New Zealand Climate Environment Laboratory (NZCEL). Treatments were terminated when the petiole elongation rate (PER) in the first fully-expanded leaf reached zero. Water relations, growth responses, the expression of the white clover ACO genes, TR-ACO1 TR-ACO2 and TR-ACO3 and the accumulation of two of the corresponding proteins, TR-ACO1 and TR-ACO2, were then examined. The soil water content (SWC) and leaf water potential (LWP) measured in both varieties and in both water deficit treatments declined progressively. The rate of decline in SWC and LWP was slower in the Tienshan ecotype with no difference between the NPS and PS treatments. However, the LWP in the Tienshan ecotype at the point at which the PER ceased was less negative (ca. -1.4 MPa) compared to Kopu (ca. -1.7 MPa). In addition, the decline in the PER differed between NPS- and PS-treated Kopu. In the NPS-treated Kopu, the PER was maintained at a high rate when plants were exposed to SWC above 18%, but declined sharply as the SWC declined further. However, in the PS-treated Kopu, the PER declined more progressively in a similar pattern to that determined for NPS- and PS-treated Tienshan. Expression of TR-ACO1 and accumulation of TR-ACO1 was observed in the apical structure of the stolon. As the water deficit progressed, no significant alteration in TR-ACO1 expression and TR-ACO1 protein accumulation was observed in the apical structures of both the NPS- and PS-treated Tienshan ecotype suggesting some degree of protection of the meristem tissues in this more drought-tolerant variety. However, a discernable decline in expression of TR-ACO1 and accumulation of TR-ACO1 protein was observed in the NPS-treated Kopu suggesting some degree of tissue injury in this more drought-susceptible variety. However, after the pre-stress (PS) treatment, no real changes in TR-ACO1 expression and TR-ACO1 protein accumulation were observed, in common with the observations for the NPS- and PS-treated Tienshan ecotype suggesting that meristem protection may now be occurring. The results suggest further that the pre-stress treatment of the more drought-susceptible Kopu may result in a degree of acclimation to the water deficit. For the first-fully expanded leaves, expression of two transcripts, TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein was monitored as the SWC decreased. The expression of TR-ACO2 and accumulation of TR-ACO2 decreased as the water deficit progressed in both the NPS- and PS-treated Tienshan ecotype and correlated with the decrease in PER. By contrast, in the NPS-treated Kopu, TR-ACO2 expression and TR-ACO2 protein accumulation increased, but again, after a period of pre-stress, TR-ACO2 expression and TR-ACO2 accumulation decreased, in common with the Tienshan ecotype. Again, the pre-stress treatment of the drought-susceptible Kopu may result in a degree of acclimation to the water deficit such that the responses become similar to those observed in the more drought-tolerant Tienshan ecotype. However, in both NPS- and PS-treated Tienshan and Kopu there was no significant alteration in the expression of TR-ACO3 in the first fully-expanded leaf. The expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were also observed in the second fully-expanded leaves (an older tissue). Again similar patterns in the expression of TR-ACO2 and TR-ACO3 and accumulation of TR-ACO2 protein were observed in both NPS- and PS-treated Tienshan and Kopu. In these leaves, expression of TR-ACO2 and accumulation of TR-ACO2 protein decreased as the water deficit progressed, but expression of TR-ACO3 increased as the water deficit decreased to less than 10%. These results suggest that responses of younger tissues (apical structure; first-fully expanded leaf) maybe the critical determinant for the tolerant (or otherwise) of white clover plants to water deficit. In the second part of this thesis, four ACS genes were identified from the Tienshan ecotype exposed to water deficit and designated TR-ACS-T. Three of these were similar to previously identified TR-ACS genes from Grasslands Challenge genotype 10F while the fourth was a novel gene designated TR-ACS4-T. TR-ACS4-T is 64%, 64% and 63% homologous to TR-ACS1-T, TR-ACS2-T and TR-ACS3-T, respectively in terms of nucleotide sequence. In the GeneBank database, TR-ACS4-T shares highly homology to ACC synthase sequences from a wide range of tissues including seedlings and fruit tissues, in addition to a high homology to ACS genes induced in auxin-, wounding- and ethylene-treated tissues. The pattern of TR-ACS4-T expression observed during leaf development suggests that the gene is expressed initially in the apical structures and in the newly initiated leaves, and then again in the later mature leaves and those at the onset of senescence. Expression decreases again during senescence. TR-ACS4-T expression is not altered by water deficit, but is induced by both ethylene and NAA treatment, but the auxin-induced TR-ACS4-T is mediated by ethylene treatment.

Phosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.

Phosphate (P) supply is one of the major determining factors to plant productivity, since the element affects the growth and the development of plants. In response to Pdeficiency treatment, plants display alterations in root system architecture caused by changes in primary root (PR) and lateral root (LR) length and LR density. In this thesis, the root growth of the agronomically important legume, white clover (Trifolium repens L.) was found to be slightly stimulated in terms of PR length, LR number and total LR length when plants were grown in a P-deficient media (0.01 mM orthophosphate; Pi) when compared with plants grown in a P-sufficient media (1.00 mM Pi) when using a hydroponic growth system. When plants are grown in a P-sufficient media, treatment with 100 nM exogenous 1- aminocyclopropane-1-carboxylic acid (ACC) and exogenous auxin (5 nM 1- naphthylacetic acid, NAA) resulted in significant increases in white clover PR length, LR number and LR length. However, when ethylene action or auxin transport were inhibited using 300 ppm 1-methylcyclopropene (1-MCP) and 100 nM 1-Nnaphthylphthalamic acid (NPA), respectively, root growth was significantly reduced which suggests roles for ethylene and auxin in mediating white clover root growth. To examine the effects of these hormones on plants grown in P-deficient media, 100 nM ACC treatment significantly enhanced the stimulatory effects of growth on Pdeficient media only, while exposure of plants to P-deficiency alone was sufficient to significantly neutralise the inhibitory effects of 1-MCP on root growth. Hence, exposure to P-deficiency is proposed to increase either ethylene biosynthesis or ethylene sensitivity in white clover roots. In contrast, for plants grown in P-deficient media, treatment with 5 nM NAA significantly abolished the stimulation of white clover root growth observed with P-deficiency so it is proposed that exposure to Pdeficiency increases either auxin biosynthesis or auxin sensitivity, but the 5nM NAA concentration used was too high to stimulate root growth. Using DR5p::GUS transgenic white clover, auxin activity was found in the root tips and root primordia. Using these plants, it is suggested that P-deficient treatment and ACC treatment influenced white clover root growth through an increase in auxin sensitivity. Overall, ethylene and auxin are found to be essential in mediating white clover root growth in P-sufficiency, and also in mediating root responses to P-deficiency through changes in terms of the biosynthesis and the sensitivity of these two hormones.

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2018-06-14T13:19:23Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_natalia_silveira_correa.pdf: 16569 bytes, checksum: ed2405236282a6dfb013ce0c61b1b3aa (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-06-14T20:52:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_natalia_silveira_correa.pdf: 16569 bytes, checksum: ed2405236282a6dfb013ce0c61b1b3aa (MD5) / Made available in DSpace on 2018-06-14T20:52:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) resumo_tese_natalia_silveira_correa.pdf: 16569 bytes, checksum: ed2405236282a6dfb013ce0c61b1b3aa (MD5) Previous issue date: 2017-06-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O objetivo deste trabalho foi avaliar a viabilidade, o vigor da semente e o crescimento de plantas de cornichão e trevo vermelho, provenientes de áreas da Embrapa Pecuária Sul, submetidas a diferentes doses de herbicidas pré e pós emergentes (diclosulam, bentazona e iodosulfurom-metílico), com a finalidade de identificar os herbicidas seletivos à estas forrageiras. Na primeira etapa do experimento foi avaliado o efeito do herbicida pré-emergente diclosulam no potencial fisiológico das sementes e crescimento inicial das plântulas. As sementes das forrageiras, foram semeadas em substrato previamente umedecido com solução de diclosulam nas concentrações de 0; 0,5; 1,0 e 1,5 vezes a dose registrada do produto pelo fabricante. Na segunda etapa, após o cultivo em casa de vegetação por 107 dias, foi realizada a aplicação dos pósemergentes, por meio de pulverizador costal de pressão constante em condições atmosféricas de 23º C de temperatura e 71% de umidade relativa do ar (UR). Foram utilizadas concentrações do herbicida referentes a zero; 0,5; 1,0 e 1,5 vezes a dose registrada pelo fabricante. Os resultados da primeira etapa, demostraram que as variáveis comprimento da parte aérea, comprimento de raiz e massa seca total foram alteradas negativamente pela exposição às diferentes doses de diclosulam, enquanto o potencial germinativo e a capacidade de emergir não foram prejudicadas, em ambas as espécies estudadas. Na segunda etapa os resultados obtidos mostraram maior sensibilidade das plantas de cornichão ao herbicida bentazona e das plantas de trevo vermelho ao herbicida iodosulfurom-metílico. Portanto é possível concluir que, as diferentes doses de diclosulam não comprometem o potencial germinativo, nem a capacidade de emergir das sementes de cornichão e trevo vermelho, porém, interferem negativamente no seu desenvolvimento. Já com a aplicação dos pósemergentes, o cornichão apresentou sensibilidade ao herbicida bentazona e características de tolerância ao herbicida iodosulfurom-metílico, enquanto o trevo vermelho respondeu de forma contrária, sendo mais sensível a aplicação do iodosulfurom-metílico e tolerante ao bentazona, ativando mecanismos de adaptação e permitindo o desenvolvimento da planta. / The objective of this research was to evaluate viability, seed vigor and growth of birdsfoot trefoil and red clover, plants from Embrapa Pecuária Sul, was submitted to different doses of pre and post emergent herbicides (diclosulam, bentazon and iodosulfuron-methyl), with purpose to identify herbicides selective to these forages. In the first stage of the experiment the effect of the pre-emergent herbicide diclosulam in physiological potential of seeds and initial growth of seedlings was evaluated. The seeds of this forages were sown on a substrate previously moistened with diclosulam solution at concentrations 0; 0,5; 1,0 and 1,5 times the dose registred by manufacturer. In second stage, after the cultivation in the greenhouse for 107 days, the postemergence application was carried out by a costal sprayer with constant pressure at atmospheric conditions of 23 ºC and 71% relative humidity). Herbicide concentrations used were zero; 0,5; 1,0 and 1,5 times the dose registred by manufacturer. The results of the first stage showed that the variables shoot length, root length and total dry mass were negatively altered by exposure to different doses of diclosulam, while germination potential and emergence capacity were not impaired in both species studied. In the second stage, the results obtained showed greater sensitivity of birdsfoot trefoil plants to herbicide bentazon and the red clover plants to iodosulfuron-methyl herbicide. Therefore, it is possible to conclude that, different doses of diclosulam do not compromise germination potential, neither ability to emerge from birdsfoot trefoil and red clover seeds, but negatively interfere in its development. With the post-emergence application, the birdsfoot trefoil showed sensitivity to herbicide bentazon and characteristics of tolerance to herbicide iodosulfuron-methyl, while the red clover responded in a contrary way, being more sensitive the application of the herbicide iodosulfuron-methyl and tolerant to bentazon, activating mechanisms of adaptation and allowing plant development.