Made available in DSpace on 2014-06-11T19:23:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-10-05Bitstream added on 2014-06-13T18:50:12Z : No. of bitstreams: 1 locardi_b_me_rcla.pdf: 766585 bytes, checksum: 800fa64d7757a908188ffc932a3ac9b6 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A diversidade de estratégias reprodutivas e de germinação de sementes da flora do cerrado brasileiro é consequência da heterogeneidade fisionômica e da sazonalidade. Essas estratégias são adaptativas para o momento onde temperatura e umidade permitam o estabelecimento de uma nova geração de plantas. O momento de dispersão e o controle fisiológico da aptidão a germinar são estratégias para a sincronização com a estação chuvosa. A sincronização fisiológica é feita pela flutuação da dormência a partir de sinais ambientais, e seus principais sinalizadores são os hormônios vegetais ABA (inibidor da germinação e agente do aprofundamento da dormência) e GA (promotor da germinação). Por nível de dormência entende-se um estado de estabilidade da semente onde certas condições ambientais podem promover a germinação, inibi-la ou ainda aprofundar esse nível a um estado sensível a outras condições ambientais. Buscamos compreender a influencia da variação da temperatura e umidade de solo pos dispersão na germinação de sementes X. aromatica, B. variabilis e V. tucanorum. Para inferir a flutuação hormonal causada pela sazonalidade, a sensibilidade das sementes a GA4+7 e fluridona e água foi acompanhada pós enterrio. As áreas experimentais foram duas, a Reserva Experimental de Mogi-Guaçu e o Jardim Experimental da UNESP Rio Claro. Os dados de temperatura, umidade do solo foram registrados a cada na profundidade de 5 cm, mesma do enterrio. A viabilidade inicial das sementres das três espécies é acima de 75%, mas a germinabilidade de B. variabilis é de 30%. As sementes de V. tucanorum e B. variabilis têm embriões morfologicamente maduros já as sementes de X. aromatica são morfo-fisiologicamente dormentes. Sendo pós-dispersão, sensíveis a GA e perdendo a sensibilidade com o aprofundamento... / A great number of reproduction and germination strategies are found in plants of the neotropical savannas (Brazilian Cerrados) due to its physiognomic diversity and seasonality. That allows a new generation establishment when temperature and moisture are less harsh than the dry winter. Dispersion time and physiological control of germination ability are regular strategies used to synchronize it with rainy season. Synchronization is regulated by changes on dormancy level by environmental cues, mainly controlled by ABA (germination inhibitor and dormancy agent) and GA (germination promoter). Dormancy level is a seed stable state in which environmental cues can promote or inhibit germination or also change it into a different level more sensitive to other environmental condition. Observing X. aromatica, B. variabilis and V. tucanorum seeds morphology, germination and viability, after dispersion and burial and incubated on water, GA4+7 .Its in situ germination was observed at 2 burial sites (Reserva Experimental de Mogi-Guaçu (MG) and Jardim Experimental da UNESP Rio Claro(JD)) after dispersion, combined with soil temperature and moisture at 5cm deep, and environmental data we search to comprehend how seasonality affects the seed’s dormancy levels, germination, mortality and hormonal sensitivity. All the three species have over 75% viable embryos, although B. variabilis germinability is less than 30%. X. aromatica seeds are morpho physiolocally dormant, being sensible to GA after dispersion and losing this sensitivity by dormancy enhanced by the burials and alternating high amplitude of temperature followed by days with amplitude lower than 10°C break these deep dormancy and on moisture soil, allows the embryo development and germinate. Lower moisture content delays the embryo... (Complete abstract click electronic access below)

Orientador: Massanori Takaki / Banca: Edvaldo Aparecido Amaral da Silva / Banca: Antonio Carlos Silva de Andrade / Resumo: A diversidade de estratégias reprodutivas e de germinação de sementes da flora do cerrado brasileiro é consequência da heterogeneidade fisionômica e da sazonalidade. Essas estratégias são adaptativas para o momento onde temperatura e umidade permitam o estabelecimento de uma nova geração de plantas. O momento de dispersão e o controle fisiológico da aptidão a germinar são estratégias para a sincronização com a estação chuvosa. A sincronização fisiológica é feita pela flutuação da dormência a partir de sinais ambientais, e seus principais sinalizadores são os hormônios vegetais ABA (inibidor da germinação e agente do aprofundamento da dormência) e GA (promotor da germinação). Por nível de dormência entende-se um estado de estabilidade da semente onde certas condições ambientais podem promover a germinação, inibi-la ou ainda aprofundar esse nível a um estado sensível a outras condições ambientais. Buscamos compreender a influencia da variação da temperatura e umidade de solo pos dispersão na germinação de sementes X. aromatica, B. variabilis e V. tucanorum. Para inferir a flutuação hormonal causada pela sazonalidade, a sensibilidade das sementes a GA4+7 e fluridona e água foi acompanhada pós enterrio. As áreas experimentais foram duas, a Reserva Experimental de Mogi-Guaçu e o Jardim Experimental da UNESP Rio Claro. Os dados de temperatura, umidade do solo foram registrados a cada na profundidade de 5 cm, mesma do enterrio. A viabilidade inicial das sementres das três espécies é acima de 75%, mas a germinabilidade de B. variabilis é de 30%. As sementes de V. tucanorum e B. variabilis têm embriões morfologicamente maduros já as sementes de X. aromatica são morfo-fisiologicamente dormentes. Sendo pós-dispersão, sensíveis a GA e perdendo a sensibilidade com o aprofundamento... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: A great number of reproduction and germination strategies are found in plants of the neotropical savannas (Brazilian Cerrados) due to its physiognomic diversity and seasonality. That allows a new generation establishment when temperature and moisture are less harsh than the dry winter. Dispersion time and physiological control of germination ability are regular strategies used to synchronize it with rainy season. Synchronization is regulated by changes on dormancy level by environmental cues, mainly controlled by ABA (germination inhibitor and dormancy agent) and GA (germination promoter). Dormancy level is a seed stable state in which environmental cues can promote or inhibit germination or also change it into a different level more sensitive to other environmental condition. Observing X. aromatica, B. variabilis and V. tucanorum seeds morphology, germination and viability, after dispersion and burial and incubated on water, GA4+7 .Its in situ germination was observed at 2 burial sites (Reserva Experimental de Mogi-Guaçu (MG) and Jardim Experimental da UNESP Rio Claro(JD)) after dispersion, combined with soil temperature and moisture at 5cm deep, and environmental data we search to comprehend how seasonality affects the seed's dormancy levels, germination, mortality and hormonal sensitivity. All the three species have over 75% viable embryos, although B. variabilis germinability is less than 30%. X. aromatica seeds are morpho physiolocally dormant, being sensible to GA after dispersion and losing this sensitivity by dormancy enhanced by the burials and alternating high amplitude of temperature followed by days with amplitude lower than 10°C break these deep dormancy and on moisture soil, allows the embryo development and germinate. Lower moisture content delays the embryo... (Complete abstract click electronic access below) / Mestre

A cana-de-açúcar é uma das principais e dos mais antigos cultivos do Brasil, ocupando lugar de destaque na economia do país devido à produção de açúcar e etanol. Atualmente, São Paulo é o principal estado produtor, impulsionando a economia brasileira devido as suas grandes áreas plantadas. O uso de reguladores vegetais vem se tornando uma prática rotineira, objetivando maximizar o potencial produtivo das culturas, promovendo melhorias na qualidade, otimizando os resultados agroindustriais e econômicos. Giberelina é um hormônio conhecido como promotor de crescimento e alongamento das plantas. O ethephon é um maturador vegetal muito utilizado na cultura da cana, promovendo a liberação de etileno quando em contato com o tecido vegetal. O objetivo do trabalho foi estudar a eficácia dos reguladores vegetais giberelina (GA) e ethephon (CEPA) aplicados em diferentes fases do desenvolvimento inicial, na morfologia e aspectos tecnológicos da cana-de-açúcar. Para isso foram efetuados quatro experimentos. As concentrações aplicadas de GA (0; 10; 25; 50 e 75 mg L-1) e CEPA (0; 225; 450; 900 e 1000 mg L-1), foram iguais nos dois primeiros experimentos. O primeiro experimento foi realizado no Horto Experimental do Departamento de Ciências Biológicas da ESALQ/USP, com a pulverização dos toletes, com dez repetições e o segundo foi realizado na Estação Experimental da Syngenta em Holambra com a pulverização de plantas jovens (na soqueira), com quatro repetições, sendo que os parâmetros biométricos foram avaliados quinzenalmente até aos 90 dias após o plantio (DAP). O terceiro experimento foi efetuado em casa de vegetação no Horto Experimental do Departamento de Ciências Biológicas da ESALQ/USP, sendo que para a avaliação dos parâmetros tecnológicos no início do desenvolvimento, pulverizaram-se toletes com GA 50 mg L-1 e CEPA 900 mg L-1 além do controle, utilizando-se de cinco repetições. As amostras foram coletadas e congeladas, para posteriores análises tecnológicas. No quarto experimento, avaliou-se a aplicação dos reguladores vegetais in vitro, onde se aplicou GA (0; 2,5; 5,0; 7,5 e 10 mg L-1) e CEPA (0; 25; 50; 100; 200 mg L-1), com 5 repetições. Os resultados foram submetidos a análise de variância pelo teste F, e comparados pelo teste de Tukey à 5% de probabilidade. No primeiro experimento, a aplicação de GA nos toletes reduziu de forma geral o desenvovimento inicial das plantas, enquanto os tratamentos com CEPA apresentaram resultados promissores ao desenvolvimento inicial, quando os parâmetros avaliados foram os perfilhos. No segundo experimento, a aplicação de GA em soqueira aumentou a altura da planta, sendo que CEPA retardou a altura das plantas e incrementou o número de perfilhos. No terceiro experimento, nas avaliações tecnológicas, GA e CEPA afetaram temporariamente os teores de açúcares totais produzidos nas folhas. No colmo, CEPA e GA afetaram de forma geral os parâmetros tecnológicos, reduzindo as atividades das invertases e os açúcares redutores e totais e No quarto experimento, a aplicação in vitro de GA, reduziu o número de perfilhos, enquanto aplicação de CEPA incrementou o número de perfilhos e reduziu a altura das plantas. / Sugarcane is one of the major and oldest crops in Brazil, taking a prominent place in the economy because of the production of sugar and ethanol. Currently, Sao Paulo is the main producing state, boosting the Brazilian economy due to its large planted areas. The use of plant regulators has become a routine practice, aiming to maximize the yield potential of crops, improving quality, optimizing results for the agroindustry and economy. Gibberellin is a plant hormone known as a regulator of plant growth and elongation. Ethephon is a growth regulator widely used in maturing sugarcane cultivation, allowing the release of ethylene when in contact with the plant tissue. This work aimed to evaluate the effectiveness of plant regulators gibberellin (GA) and ethephon (CEPA), applied at different stages of early development, on the morphology and technological aspects of sugarcane. To that end, four experiments were performed. The concentrations of GA (0; 10; 25; 50 and 75 mg L-1) and CEPA (0; 225; 450; 900 and 1000 mg L-1), were equal in the first two experiments. The first experiment was conducted in the Experimental Garden of the Department of Biological Sciences, ESALQ / USP, by spraying of cuttings, with ten repetitions. The second was conducted at Syngenta\'s experimental station in Holambra, by spraying the young plants (at stumps) with four replicates, and biometric parameters were measured fortnightly until 90 days after planting (DAP). The third experiment was conducted in a greenhouse at the Experimental Garden of the Department of Biological Sciences, ESALQ / USP, and for the evaluation of technological parameters in early development, cuttings were sprayed with GA 50 mg L-1 and CEPA 900 mg L-1 besides the control, using five replicates. Samples were collected and frozen for later technological analysis. In the fourth experiment, the application of plant regulators in vitro was evaluated, where GA (0; 2.5; 5.0; 7.5 and 10 mg L-1) and CEPA (0; 25; 50; 100; 200 mg L-1) were applied with five replicates. The results were submitted to variance analysis by F test and compared by Tukey test at 5% probability. In the first experiment, the application of GA reduced, in general terms, the initial development of plants whereas treatments with CEPA had promising results in early development, regarding the tillering. In the second experiment, the application of GA increased plant height, while the CEPA retarded plant height and increased the number of tillers. In the third experiment in technological assessments, GA and CEPA temporarily affected the contents of total sugars in the leaves. In the stem, GA and CEPA affected, in general, technological parameters, reducing the activities of invertase as well as those of the reducing and total sugars. In the fourth experiment, the application of GA in vitro decreased the number of tillers, while the treatment with CEPA increased the number of tillers and delayed the plant height.

As espécies selvagens relacionadas ao tomateiro (Lycopersicon esculentum Mill. Syn. Solanum lycopersicum L) em latitudes do Sul do Equador ao norte do Chile. Estas condições ambientais contrastantes possibilitaram o aparecimento de grande diversidade genética dentre estas espécies. Lycopersicon cheesmanii f .minor (Hook.f.) C. H. Hill. Syn. Solanum galapagense S. Darwin & Peralta é endêmica das Ilhas Galápagos e possui características peculiares, como porte reduzido, dormência de sementes e folhas bastante recortadas. Estas características também estão presentes em mutantes de tomateiro com deficiência no hormônio giberelina (GA). Mutações em GA como gib1, gib2, gib3 (deficientes) e procera (resposta constitutiva) são amplamente conhecidas em tomateiro. Já os alelos Sp (Self Pruning) e Pts (Petroselinum), presentes em S. galapagense, alteram altura e recorte foliar, respectivamente. Visando entender a natureza destas variações genéticas naturais, foram feitos cruzamentos e retrocruzamentos sucessivos de S. galapagense com a cultivar miniatura de tomateiro Micro-Tom (MT), onde tentou se isolar plantas segregando para características presentes no parental selvagem (dormência, nanismo e maior recorte foliar). Na geração BC1F2 foram selecionados indivíduos com porte menor que MT e folhas com bordos muito recortados. Sementes BC1F3 apresentaram taxa de germinação de 47,6 %, contrastando com o valor 94,5 % apresentado por MT. Entretanto, após aplicação de 100 µM de GA3 a taxa de germinação de BC1F3 foi elevada para 72 %. Em análise de curvas de dose-resposta a GA, as plantas BC1F3 apresentaram menor porte que MT, sendo que este nanismo não foi completamente revertido aplicando-se GA. Em gerações avançadas de retrocruzamentos, constatou-se que a dormência de sementes foi perdida durante as introgressões. Tal constatação leva a crer que o principal componente da dormência de S. galapagense não está ligado ou é efeito pleiotrópico dos genes que respondem pelo seu nanismo ou recorte foliar. A análise de segregação de 278 plantas BC4F2 mostrou que o principal componente do nanismo de S. galapagense segrega na proporção 3:1, sendo a mutação recessiva presente em S. galapagense denominada galapagos dwarf (gdw). Plantas quase isogênicas a MT (geração BC6Fn) foram obtidas carregando os alelos Pts, Sp e gdw. Experimentos comparando-as com MT confirmaram que o alelo Pts aumenta pronunciadamente o recorte foliar de tomateiro e diminui ligeiramente a germinação e o porte das plantas. Tal observação está de acordo com a descoberta recente de que PTS codifica para um gene da classe KNOX, que podem estar envolvidos com GA. Em cominação com Pts, esse alelo parece ter efeito discreto no recorte foliar, mas somente em combinação com Pts. Surpreendentemente, sementes Sp tiveram germinação precoce, comparadas com MT (sp). Já o alelo gdw não mostrou ter efeito na germinação, mas confirmou afetar a altura e o recorte foliar. Esses resultados evidenciam que porte reduzido e folhas bastante recortadas de S. galapagense podem ser atribuídos principalmente a gdw e Pts. A dormência parece ser controlada por outro(s) gene(s) ainda desconhecido(s). Como o novo gene descoberto, GDW, não afeta a germinação, é pouco provável que esteja ligado a GA, podendo ser uma nova classe de genes controlando nanismo. / The tomato (Lycopersicon esculentum Mill. Syn. Solanum lycopersicum L) related wild species evolved into a wide range of latitudes, from the southern of Ecuador to the northern of Chile. These contrasting environment conditions allowed the emergence of great genetic diversity among these species. Lycopersicon cheesmanii f. minor (Hook.f.) C. H. Hill. Syn. Solanum galapagense S. Darwin & Peralta is endemic in the Galapagos Islands and has characteristics such as small plant size, dormant seeds and profusely divided leaves, being these characteristics also common in tomato gibberellin (GA) mutants. GA mutants such as gib1, gib2, gib3 (deficient), and procera (constitutive) are widely known in tomato. In addition to the afore mentioned mutations, the alleles Sp (Self Pruning) and Pts (Petroselinum), present S. galapagense, lead to changes in plant height and leaf architecture, respectively. Aiming at the understanding of such natural genetic variations, successive crosses and backcrosses of S.galapagense with the miniature tomato cultivar Micro-Tom (MT) were made, attempting to isolate plants segregating characteristics of the parental wild species (dormancy, dwarfism, and increased leaf indentation). In the BC1F2 generation were selected individuals shorter than MT, and with more serrated leaf margins or with more leaflets when compared to MT. BC1F3 seeds showed germination rates of only 46.7 %, contrasting with the 94.5% of MTs germination. However, after the application of 100 µM GA, the rate of germination of BC1F3 was increased to 72 %. Analysis GA dose-responses showed that the BC1F3 plants displayed smaller sizes than the MT plants, and this dwarfism was not completely reversed by GA applications. In advanced backcross generations, it was found that the dormancy of seeds was lost during the process of introgression, since the selection was not for this trait, but only for plant with small size and/or more divided leaves. This finding suggests that the main component of dormancy of S.galapagense is not connected to or is not a pleiotropic effect of the genes for dwarfism and leaf shape. The analyzes of the segregation of 278 BC4F2 plants, already harboring the alleles sp and pts from MT, showed that the main component of the dwarfism of S. galapagense segregates in the proportion 3:1, and then, the recessive mutation present in this specie was named galapagos dwarf (gdw). Plants nearly isogenic to MT (BC6Fn generation) were obtained carrying the alleles Pts, Sp and gdw. Experiments comparing these plants with MT confirmed that the allele Pts produces more divided leaf and leaflets, and showed that this allele has a slight effect in reducing seed germination and plant size. This observation is consistent with the recent discovery that Pts codes for a gene from the KNOX class, which may be involved with the GA hormone. The Sp allele had no direct effect on plant height, but only indirectly due to its indeterminate growth. This allele seems to have a slight effect on leaf shape, but only in combination with Pts. Surprisingly, Sp seeds had an early germination when compared to MT (sp). On the other hand, the allele gdw did not show any effect on germination, but confirmed to affect plant height and leaf architecture. Taken together, these results showed that the reduced plant size and the profusely divided leaves of S. galapagense can be attributed mainly to the effect of Pts and gdw alleles. Regarding seed dormancy, this trait appears to be controlled by other(s) gene(s), yet unknown, although Pts also contributes to this characteristic. Since the new gene that we discovered, GDW, does not affect the germination, it is unlikely that it can be linked the GA hormone, but it may represent a new class of genes controlling an important agronomic trait, the dwarfism.

Plant reproductive development begins when vegetative shoot apical meristems change their fate to inflorescence meristems which develop floral meristems on the flanks. This process of meristem fate change and organ development involves regulated activation and/or repression of many cell fate determining factors that execute down-stream gene expression cascades. Flowers are formed when floral organs are specified on the floral meristem in four concentric whorls. In the model dicot plant Arabidopsis, the identity and pattern of floral organs is determined by combined actions of MADS-domain containing transcription factors of the classes A, B, C, D and E. Rice florets are produced on a compact higher order branch of the inflorescence and have morphologically distinct non-reproductive organs that are positioned peripheral to the male and female reproductive organs. These unique outer organs are the lemma and palea that create a closed floret internal to which are a pair of lodicules that are asymmetrically positioned fleshy and reduced petal-like organs. The unique morphology of these rice floret organs pose intriguing questions on how evolutionary conserved floral meristem specifying and organ fate determining factors bring about their distinct developmental functions in rice. We have studied the functions for two rice MADS-box proteins, OsMADS2 and OsMADS1, to understand their role as master regulators of gene expression during rice floret meristem specification and organ development. OsMADS2; a transcriptional regulator of genes expression required for lodicule development Arabidopsis B-function genes AP3 and PI are stably expressed in the whorl 2 and 3 organ primordia and they together with other MADS-factors (Class A+E or C+E) regulate the differentiation of petals and stamens (Jack et al, 1992; Goto and Meyerowitz, 1994). Rice has a single AP3 ortholog, SPW1 (OsMADS16) but has duplicated PI-like genes, OsMADS2 and OsMADS4. Prior studies in our lab on one of these rice PI-like genes OsMADS2 showed that it is needed for lodicule development but is dispensable for stamen specification (Kang et al., 1998; Prasad and Vijayraghavan, 2003). Functional divergence between OsMADS2 and OsMADS4 may arise from protein divergence or from differences in their expression patterns within lodicule and stamen whorls. In this study, we have examined the dynamic expression pattern of both rice PI-like genes and have examined the likelihood of their functional redundancy for lodicule development. We show OsMADS2 transcripts occur at high levels in developing lodicules and transcripts are at reduced levels in stamens. In fully differentiated lodicules, OsMADS2 transcripts are more abundant in the distal and peripheral regions of lodicules, which are the tissues that are severely affected in OsMADS2 knock-down florets (Prasad and Vijayraghavan, 2003). The onset of OsMADS4 expression is in very young floret meristems before organ primordia emergence and this is expressed before OsMADS2. In florets undergoing organogenesis, high level OsMADS4 expression occurs in stamens and carpels and transcripts are at low level in lodicules (Yadav, Prasad and Vijayraghvan, 2007). Thus, we show that these paralogous genes differ in the onset of their activation and their stable transcript distribution within lodicules and stamens that are the conserved expression domains for PI-like genes. Since the expression of OsMADS4 in OsMADS2 knock-down florets is normal, our results show OsMADS2 has unique functions in lodicule development. Thus our data show subfunctionalization of these paralogous rice PI-like genes. To identify target genes regulated by OsMADS2 that could contribute to lodicule differentiation, we have adopted whole genome transcript analysis of wild-type and dsRNAiOsMADS2 panicles with developing florets. This analysis has identified potential down-stream targets of OsMADS2 many of which encode transcription factors, components of cell division cycle and signalling factors whose activities likely control lodicule differentiation. The expression levels of few candidate targets of OsMADS2 were examined in various floret organs. Further, the spatial expression pattern for four of these down-stream targets of OsMADS2 was analysed and we find overlap with OsMADS2 expression domains (Yadav, Prasad and Vijayraghvan, 2007). The predicted functions of these OsMADS2 target genes can explain the regulation of growth and unique vascular differentiation of this short fleshy modified petal analog. OsMADS1, a rice E-class gene, is a master regulator of other transcription factors and auxin and cytokinin signalling pathways In Arabidopsis four redundant SEPALLATA factors (E-class) are co-activators of other floral organ fate determining MADS-domain factors (classes ABCD) and thus contribute to floral meristem and floral organ development (Krizek and Fletcher, 2005). Among the grass-specific sub-clade of SEP-like genes, rice OsMADS1 is the best characterized. Prior studies in our lab showed that OsMADS1 is expressed early throughout the floret meristem before organ primordia emergence and later is restricted to the developing lemma and palea primordia with weak expression in carpel (Prasad et al, 2001). Stable expression continues in these floret organs. OsMADS1 plays critical non-redundant functions to specify a determinate floret meristem and also regulates floret organ identities (Jeon et al., 2000; Prasad et al, 2001; 2005; Agarwal et al., 2005; Chen et al., 2006). In the present study, we have adopted two different functional genomic approaches to identify genes down-stream of OsMADS1 in order to understand its mechanism of action during floret development. We have studied global transcript profiles in WT and dsRNAiOsMADS1 panicles and find OsMADS1 is a master regulator of a significant fraction of the genome’s transcription factors and also a number of genes involved in hormone-dependent cell signalling. We have validated few representative genes for transcription factors as targets regulated by OsMADS1. In a complementary approach, we have determined the consequences of induced-ectopic over-expression of a OsMADS1:ΔGR fusion protein in shoot apical meristems of transgenic plants. Transcript levels for candidate target genes were assessed in induced tissues and compared to mock-treated meristems and also with meristems induced for OsMADS1:ΔGR but blocked for new protein synthesis. These analyses show that OsMADS55 expression is directly regulated by OsMADS1. Importantly, OsMADS55 is related to SVP that plays an important role in floral transition and floral meristem identity in Arabidopsis. OsHB3 and OsHB4, homeodomain transcription factors, with a probable role in meristem function, are also directly regulated by OsMADS1. The regulation of such genes by OsMADS1 can explain its role in floret meristem specification. In addition to regulating other transcription factors, OsMADS1 knock-down affects expression of genes encoding proteins in various steps of auxin and cytokinin signalling pathways. Our differential expression profiling showed OsMADS1 positively regulates the auxin signalling pathway and negatively regulates cytokinin mediated signalling events. Through our induced ectopic expression studies of OsMADS1:ΔGR, we show OsMADS1 directly regulates the expression of OsETTIN2, an auxin response transcription factor, during floret development. Overall, we demonstrate that OsMADS1 modulates hormonal pathways to execute its functions during floret development on the spikelet meristems. Functional studies of OsMGH3; an auxin-responsive indirect target of OsMADS1 To better understand the contribution of auxin signalling during floret development, we have functionally characterized OsMGH3, a down-stream indirect target of OsMADS1, which is a member of the auxin-responsive GH3 family. The members of this family are direct targets of auxin response factors (ARF) class of transcription factors. GH3-proteins inactivate cellular auxin by conjugating them with amino acids and thus regulate auxin homeostasis in Arabidopsis (Staswick et al., 2005). OsMGH3 expression in rice florets overlaps with that of OsMADS1 (Prasad et al, 2005). In this study, we have demonstrated the consequences of OsMGH3 over-expression and knock-down. The over-expression of OsMGH3 during vegetative development causes auxin-deficient phenotypes such as dwarfism and loss of apical dominance. Its over-expression in developing panicles that was obtained by driving its expression from tissue-specific promoters created short panicles with reduced branching. The latter is a phenotype similar to that observed upon over-expression of OsMADS1. In contrast, the down-regulation of endogenous OsMGH3 through RNA-interference produced auxin over-production phenotypes such as ectopic rooting from aerial nodes. Knock-down of OsMGH3 expression in florets affected carpel development and pollen viability both of which affect floret fertility. Taken together, this study provides evidence for the importance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development. Our analysis of various conserved transcription factors during rice floret development suggest that factors like OsMADS2, OsMADS4 and OsMADS1 are master regulators of gene expression during floret meristem specification and organ development. The target genes regulated by these factors contribute to development of morphologically distinct rice florets.

En esta tesis doctoral se estudia el modo de vida tritrófico del hongo nematófago Pochonia chlamydosporia utilizando técnicas "multiómicas". Pochonia chlamydosporia (= Metacordyceps chlamydosporia) (Goddard) Zare y Gams es un hongo nematófago usado para el control de nematodos agalladores de la raíz (Meloidogyne spp.) (Forghani and Hajihassani, 2020), entre otros. P. chlamydosporia se distribuye por todo el mundo y tiene un modo de vida tritrófico, pudiendo también adoptar estilos de vida endófito y saprófito. El mecanismo que utiliza P. clamydosporia para infectar huevos de nematodo comprende la desacetilación de la quitina de su pared celular a quitosano para facilitar su degradación por quitosanasas (Aranda-Martinez et al., 2016). El quitosano es un biopolímero derivado de la quitina que también se encuentra en el exoesqueleto de artrópodos y crustáceos. El genoma de P. chlamydosporia codifica un elevado número de quitosanasas, gracias a las cuales es resistente a quitosano y puede utilizarlo como fuente de nutrientes (Palma-Guerrero et al., 2010). Ambos pueden combinarse para el control de plagas. En este trabajo de tesis doctoral se pretende estudiar mediante metabolómica, transcriptómica y genómica el modo de vida tritrófico de P. chlamydosporia añadiendo quitosano, para determinar los mecanismos de interacción del hongo en ese entorno. En último término, se pretende sentar las bases para desarrollar un sistema para reducir plagas y enfermedades de forma sostenible.

PhD (AGR) (Animal Science) / Department of Animal Science / Horticultural by-products such as seed and nut oil residues have the potential to replace conventional protein and energy sources in diets for ruminants. The objective of the study was to evaluate the nutritive value of Macadamia oil cake and Baobab seed cake as nutrient supplements for feedlot animals. Several experiments were carried-out to evaluate the nutritive value of Macadamia oil cake and Baobab seed cake as feed for ruminants. The nutrient composition of the cakes were determined using both proximate and modern methods of analysis of feeds and a t-test was used to compare the means of Macadamia oil cake and Baobab seed cake. The in sacco technique was used to determine the rumen degradability parameters of dry matter, crude protein and Amino acids of the cakes. This experiment was conducted using a completely randomized design. A three step-in vitro technique was used to conduct a post ruminal digestibility trial and the study was arranged in a completely randomized design. An apparent digestibility trial was conducted using metabolism cages and fecal bags to avoid the mixing of urine and faeces and the experiment was carried out as a completely randomized design arranged in a 2 x 2 factorial. A growth trial was conducted using a completely randomized block design arranged in a 2x2 factorial with two protein supplements and two inclusion levels as factors and blocked by sex of the lambs. The two cakes had similar (P>0.05) dry matter, fat, hemicellulose and gross energy contents. Baobab seed cake had significantly (P<0.05) higher ash, crude protein, acid detergent lignin and nitrogen free extract than macadamia oil cake. Macadamia oil cake had significantly (P<0.05) higher crude fiber, acid detergent fiber, neutral detergent fiber and cellulose concentrations. Baobab seed cake had significantly (P<0.05) higher calcium, magnesium, potassium, phosphorus (macroelements), zinc, and copper than macadamia oil cake. Macadamia oil cake was significantly (P<0.05) higher in manganese and iron contents than Baobab seed cake. Sodium content was not significantly (P>0.05) different between the two cakes. Baobab seed cake had more (Tryptophan, Cysteine, Arginine, Aspartic acid, Glutamic acid, Valine, Phenylalanine, Isoleucine, Leucine) Amino acids which were significantly (P<0.05) higher in quantity than macadamia oil cake with the two cakes having similar (P>0.05) remaining Amino acids contents. Generally Macadamia oil cake had more (P<0.05) saturated and mono-unsaturated fatty acids than Baobab seed cake whereas Baobab seed cake had more (P<0.05) poly-unsaturated fatty acids. v The in sacco rumen degradability characteristics of baobab seed cake and macadamia oil cake were estimated for dry matter and crude protein of the cakes. Macadamia oil cake had significantly (P<0.01) higher a value for dry matter than boabab seed cake. Baoaba seed cake had significantly (P<0.01) higher a value for crude protein than macadamia oil cake. the b, c, and a+b values for dry matter of both Baobab seed cake and Macadamia oil cake were not significantly (P>0.05) different form each other. However, the potential degradability (a+b) value for crude protein of baobab seed cake was significantly (P<0.01) higher than that of macadamia oil cake. The b and c constants of the two cakes for crude protein were not significantly (P>0.05) different from each other. The ED (Effective degradability) values calculated at three outflow rates (0.02, 0.05, 0.08) were estimated for dry matter and crude protein of Baobab seed cake and macadamia oil cake. The ED of the two cakes calculated at 0.02 outflow rate did not significantly (P>0.05) differ from each other. However, baobab seed cake had significantly (P<0.05) higher ED value at outflow rate of 0.05 whereas macadamia oil cake had significantly (P<0.05) higher ED value calculated at outflow rate of 0.08. The ED values of baobab seed cake for crude protein calculated at the three outflow rate were significantly (P<0.01) higher than those of macadamia oil cake. The two cakes did not significantly (P>0.05) differ in dry matter ruminal degradability but baobab seed cake had significantly (P<0.05) higher ruminal crude protein disappearance from 16 to 72 hours of incubation than macadamia oil cake. The in situ Amino acid degradation was determined on 0, 12, 16, 24, and 48 hour of ruminal incubation of the cakes’ samples and generally they were significant (P<0.05) differences for both cakes according to different incubation periods. The 3-step in vitro digestibility trial revealed that baobab seed cake had significantly (P<0.05) higher dry matter, crude protein and Amino acids digestibility values than macadamia oil cake. Apparent digestibility study revealed that nutrient intake, faecal and urine outputs, and digestibility of nutrients were not significantly (P>0.05) different between baobab seed cake and macadamia oil cake. However, lambs on 10% macadamia cake having retained significantly (P<0.05) more nitrogen than the lambs on 15% macadamia oil cake, 10% and 15% baobab seed cakes diets. However, the inclusion of either baobab seed cake or macadamia oil cake at 10% or 15% in the diets of lambs did not significantly (P>0.05) affect the DOMR, microbial protein yield and purine derivates output. For the growth trial the lambs were offered four diets formulated to contain 10% MOC (control), 15% MOC, 10 BSC and 15% BSC. The inclusion of 15% MOC and 10% BSC in the diets of lambs did not significantly (P>0.05) affect their final body weight, total weight gain, average daily feed intake, average daily weight gain, warm and cold carcass masses between these two vi groups. The inclusion of 10% MOC and 15% BSC in the diets of lambs did not significantly affect average daily feed intake and animal performance between these two groups. However, lambs on 10% MOC and 15% BSC had significantly (P<0.05) higher average daily feed intake and animal performance compared to lambs on 15% MOC diet. The feed conversion efficiency and of the lambs in the four diets were not significantly (P>0.05) different. The dressing percentage, carcass length, neck weight, fat thickness, body weight thickness and rib eye area of the carcasses of lambs in this trial did not differ significantly (P>0.05) except for spleen and skin with lambs on 10% BSC diets having the least skin weight and lambs on 10% MOC diet having heaver spleens. Generally BSC had higher (P<0.05) nutrient content than MOC. BSC and MOC were highly degradable in the rumen whereas BSC was higly digestible post-ruminally compared to MOC. The diets of fattening lambs formulated to include 10% or 15% of BSC or MOC as protein supplements did not affect the apparent digestibility of the diets. Macadamia oil cake can be incomporated in the diet of finishing lambs as a protein supplement at 10% inclusion level without compromising the growth and carcass characteristics of the lambs. Baobab seed cake as a protein supplement can be included in the diet of finishing lambs at up to 15% inclusion level without deleteriously compromising on the growth and carcass characteristics of the animals. ______________________________________ / NRF

<p dir="ltr">Deciduous leaf habits have evolved multiple times across many lineages in response to stresses like drought, cold, or darkness. This short, seasonal leaf lifespan allows trees to invest in photosynthesis during prime conditions and retreat to dormancy to survive less favorable conditions. The consequence of short leaf lifespan is that trees must perform an entire year's carbon capture into 6-8 months. This leads to leaves that are cheaper to produce than longer lived evergreen counterparts. As soon as challenging conditions have passed the leaves of deciduous trees expand rapidly; and this expansion has huge impacts on local ecosystems. Other plants like spring ephemerals have evolved to complete the majority of their life cycle before the upper canopy closes off. During the summer, deciduous leaves gather huge amounts of carbon for the trees to survive their dormancy. Finally, as the trees prepare to enter dormancy, nutrients are withdrawn from leaves as the chlorophyll is metabolized, causing them to transition from bright green to shades of red and yellow. In addition to other plants, people find the annual process of renewal on bud burst and tragic decline during senescence fascinating and culturally important. The aim of my thesis is to expand our understanding of winter deciduous leaves through every major stage of development, as well as to investigate how this process may shift due to climate change.</p>

La croissance d’un organisme multicellulaire pour atteindre une taille bien définie, nécessite une coordination de la prolifération cellulaire, de l’expansion et de la différentiation cellulaire ainsi que de la mort cellulaire. Ces processus sont sous l’influence de l’état nutritionnel de l’organisme, les conditions de son environnement et des signaux hormonaux. Translationally controlled tumor protein (TCTP) est un facteur essentiel à la croissance des plantes et des animaux. La protéine TCTP de plante contrôle la croissance mitotique, tandis que la protéine TCTP animale contrôle la croissance mitotique et post-mitotique. Une voie importante dans la régulation de la croissance en réponse aux nutriments est la voie Target of Rapamycin (TOR). Chez la Drosophile, il a été montré que dTCTP serait un régulateur positif en amont de TOR. Au cours de ma thèse, j’ai étudié le lien entre TCTP et la voie TOR, afin de savoir si, comme chez les animaux, AtTCTP agit en amont de la voie TOR pour contrôler la croissance des organes. Afin de savoir si la voie TCTP était liée à l’état nutritionnel, j’ai recherché l’impact du milieu de culture sur la létalité de la mutation tctp. J’ai ensuite caractérisé l’impact de la mutation tctp sur le transport et l’homéostasie de l’hormone auxine. J’ai enfin analysé pourquoi TCTP de plante ne contrôle pas la croissance post-mitotique par expansion cellulaire, contrairement à TCTP animale. Les données de la littérature montrent que chez les animaux TCTP est un activateur positif en amont de la voie TOR. Chez la plante Arabidopsis thaliana, mes données d’interactions génétiques sont en faveur d’un modèle dans lequel AtTCTP agit indépendamment de la voie TOR, contrairement de ce qu’il a été proposé chez les animaux. Chez les plantes, la perte de fonction de TCTP est associée à un retard du développement embryonnaire et à la mort. Cette létalité peut être complémentée par sauvetage des embryons sur du milieu riche en nutriments. J’ai montré que l’ajout de sucrose ou de glutamine dans le milieu de sauvetage des embryons tctp est nécessaire à leur développement. Ces données suggèrent qu’in vitro, AtTCTP n’est pas nécessaire à l’approvisionnement et à l’utilisation des nutriments sucrose, glucose ou glutamine. Dans leur ensemble, ces résultats réévaluent le rôle du régulateur de croissance TCTP en montrant que le gène AtTCTP régule la croissance mitotique indépendamment de la voie TOR et des voies de signalisation liées aux nutriments. L’observation des flux d’auxine en suivant la localisation de PIN1-GFP dans les embryons et les inflorescences du mutant tctp ne montre aucune altération par rapport au phénotype sauvage. De même, l’homeostasie de l’auxine, suivie à l’aide du rapporteur DR5::GFP n’est pas altérée dans les embryons tctp. Ceci suggère que le défaut de croissance du mutant tctp n’est pas lié à une altération du flux ou de l’homéostasie de l’auxine. La protéine TCTP de plante ne contrôle pas la croissance post-mitotique, contrairement à la protéine TCTP animale. J’ai réalisé un échange de domaines protéiques entre AtTCTP et Drosophila dTCTP. Le but était d’identifier les domaines protéiques de la protéine TCTP animale qui permettent la croissance post-mitotique. La plupart des protéines chimères étaient instables dans la Drosophile. Afin de comprendre pourquoi, j’ai réalisé du modelage par homologie et j’ai discuté la structure des chimères dans ma thèse.L’ensemble de mes résultats permet de mieux comprendre la fonction de TCTP chez les végétaux, en montrant que cette fonction s’exerce indépendamment de la voie TOR. / The growth of a multicellular organism and its size determination require the tight regulation of cell proliferation, cell differentiation, cell growth and apoptosis. These processes are influenced by the nutritional state of the organism, its environmental conditions and hormonal signals. Translationally controlled tumor protein (TCTP) is an essential regulator of growth in plants and animals. In plants it controls mitotic growth, whereas in animals, it controls mitotic and post-mitotic growth. One of the important pathways involved in the control of growth in response to nutrients is the Target of Rapamycin (TOR) pathway. In Drosophila, dTCTP was proposed to act a positive regulator upstream of TOR, although this role remains a matter of debate in the animal field.During the past 3 years of my PhD. thesis, I addressed the question whether plant TCTP acts upstream of TOR to control organ growth. I studied the impact of nutrient availability and hormones on TCTP role to control growth in plants and vice versa. Finally, I examined why plant TCTP does not control post-mitotic cell expansion growth, conversely to animal TCTP using a structure-function approach.In animals, TCTP was proposed to act as a positive activator upstream of the TOR pathway. In plants, my data support a model in which AtTCTP acts independently from the plant TOR pathway, thus in contrast to what has been proposed in animals. TCTP loss of function leads to delay of embryo development and death. Nutrient supplement rescues this embryos lethality. First, I demonstrate that embryos grown on nutrients lacking sucrose or glutamine fail to develop correctly. My data demonstrate that in vitro AtTCTP is not essential to the uptake, the use of and the response to the nutrients glucose, sucrose or glutamine. Taken together, these results reevaluate the role of AtTCTP as a growth regulator controlling mitotic growth independently from the TOR pathway and likely from nutrient related signaling pathways. Interestingly, my data also show that AtTCTP controls growth independently from auxin flux or homeostasis and that auxin-induced growth can occur without TCTP. To address why plant TCTP do not control post-mitotic growth conversely to animal counterpart, I performed protein domain swaps and created chimera proteins between Arabidopsis AtTCTP and Drosophila dTCTP. The rational was to identify protein domains that differentiate plant and animal TCTPs with regard to post-mitotic growth control. Most of chimera proteins were instable and I was unable to complement tctp loss of function in Drosophila. I performed a structure based modeling to understand this phenotype and the outcome is discussed in my PhD thesis.Altogether my results improve the understanding of plant morphogenesis by reevaluating the role of the central growth regulator TCTP.

Made available in DSpace on 2016-04-28T14:59:00Z (GMT). No. of bitstreams: 1 Cleiton Mateus Sousa.pdf: 6410770 bytes, checksum: 26a93d90d24ec7420524c2e0478aec38 (MD5) Previous issue date: 2008-07-25 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The determination of plant hormones can be an excellent tool for to study the plant development. Today, the complexity of methods, equipment and reagents of high cost limit the use with practice of routine. Some advantages of methods immunoassay can exceed those limitations, showing a potential of practical use in determination of plant hormones. The test Enzyme-Linked Immunosorbent Assay (ELISA) is utilized in detection of molecules with low molecular weight. However, the ELISA requires specific antibodies against molecules of interest, in the case, plant hormones. Today in the market there is not available antibodies for the determination of hormonal molecules. Faced with that, proposed be produced and characterize antibodies against molecules of AIA, 2ip and zeatin and subsequently, for use the detection those molecules. For that, the hormonal molecules were conjugated with protein (BSA) for subsequent immunization of chicken or rabbits. From the serum of rabbits or yolks eggs the antibodies were purified and characterized through the test of precipitation of double diffusion, SDS-PAGE and test ELISA. The antibodies that presented better results were utilized in detection of plant hormones molecules in samples of plantlets in vitro of gerbera. The test of precipitation of double diffusion revealed that the antibodies productized were capable of will detect the molecule in study. Through in the SDS-PAGE was verified that the antibodies obtained in eggs yolks of chicken presented superior purity than them obtained in serum of rabbits. From the results of the test ELISA, observed itself that the antibodies against AIA did not present potential of practical use. The detection of plant hormones in samples of tissue stayed restricted to 2ip and zeatin. The detection of those two molecules in crude extract, obtained from plantlets in vitro of gerbera, revealed that the level endogenous of zeatin was higher of level 2ip. Plantlets with six weeks after multiplication presented higher level of zeatin than plantlets with one week after multiplication. For the 2ip, that difference was not evident. The use of antibodies obtained in eggs yolks of chicken permitted the determination of zeatin and 2ip in samples plant utilizing the test ELISA. / A determina??o do n?vel hormonal end?geno pode ser uma excelente ferramenta para estudar o desenvolvimento vegetal. Atualmente, fica limitada devido a complexidade das metodologias adotadas, uma vez que demandam equipamentos e reagentes de alto custo e ainda apresentam baixo rendimento. Por outro lado, os ensaios imunoenzim?ticos possuem algumas vantagens que superam essas limita??es, demonstrando um potencial de uso pr?tico na dosagem de horm?nios vegetais. Entre os ensaios imunoenzim?ticos, destaca-se o teste Enzyme-Linked Immunosorbent Assay (ELISA), o qual vem sendo utilizado na detec??o de mol?culas com baixo peso molecular. No entanto, o teste ELISA exige anticorpos espec?ficos e que sejam capazes de reconhecer as mol?culas de interesse, no caso os horm?nios vegetais. Hoje no mercado n?o h? anticorpos dispon?veis para a determina??o das mol?culas hormonais. Diante disso, prop?s-se produzir e caracterizar anticorpos contra mol?culas de AIA, 2ip e zeatina e posteriormente, us?-los na detec??o dessas mol?culas. Para isso, as mol?culas hormonais foram conjugadas com prote?na (BSA) para posterior imuniza??o de galinhas poedeiras ou coelhos. A partir do soro de coelhos ou de gemas de ovos de galinhas os anticorpos foram purificados e caracterizados atrav?s do teste de imunodifus?o, SDSPAGE e teste ELISA. Os anticorpos que apresentaram melhores resultados foram utilizados na detec??o de mol?culas hormonais em amostras de tecidos de pl?ntulas mantidas in vitro. O teste de imunodifus?o revelou que os anticorpos obtidos foram capazes de detectarem a mol?cula em estudo. Atrav?s do SDS-PAGE verificou-se que os anticorpos obtidos em gemas de ovos de galinhas apresentaram maior pureza que os obtidos em coelhos, sugerindo que os mesmos possuem maior potencial de uso pr?tico. A partir dos resultados do teste ELISA, observou-se que os anticorpos contra AIA n?o apresentaram potencial de uso pr?tico na determina??o dessa mol?cula em amostras vegetais. Sendo assim, a detec??o de mol?culas hormonais em amostras de tecidos vegetais ficou restrita a 2ip e a zeatina. A detec??o dessas duas mol?culas em extrato bruto, obtidos a partir de pl?ntulas de g?rbera mantidas in vitro, revelou que o n?vel end?geno de zeatina foi superior ao n?vel de 2ip. Pl?ntulas com seis semanas ap?s a repicagem apresentaram maior n?vel de zeatina do que pl?ntulas rec?m repicadas, enquanto para o 2ip, essa diferen?a n?o foi evidente. O uso de anticorpos obtidos em gemas de ovos de galinhas permitiu a detec??o e quantifica??o de zeatina e 2ip em amostras vegetais utilizando o teste ELISA.