Vegetative propagation allows the amplification of selected genotypes for research, breeding, and commercial planting. However, efficient in vitro regeneration and genetic transformation remains a major obstacle to research and commercial application in many plant species. Our aims are to improve knowledge of gene regulatory circuits important to meristem organization, and to identify genes that might be useful for improving the efficiency of in vitro regeneration. In this thesis, we have approached these goals in two ways. First, we analyzed gene expression during poplar (Populus) regeneration using an AffymetrixGeneChip® array representing over 56,000 poplar transcripts. We have produced a catalog of regulated genes that can be used to inform studies of gene function and biotechnology. Second, we developed a GUS reporter system for monitoring meristem initiation using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). This provides plant materials whose developmental state can be assayed with improved speed and sensitivity. For the microarray study, we hybridized cDNAs derived from tissues of a female hybrid poplar clone (INRA 717-1 B4, Populus tremula x P. alba) at five sequential time points during organogenesis. Samples were taken from stems prior to callus induction, at 3 days and 5 days after callus induction, and at 3 and 8 days after the start of shoot induction. Approximately 15% of the monitored genes were significantly up-or down-regulated based on both Extraction and Analysis of Differentially Expressed Gene Expression (EDGE) and Linear Models for Microarray Data (LIMMA, FDR<0.01). Of these, over 3,000 genes had a 5-fold or greater change in expression. We found a very strong and rapid change in gene expression at the first time point after callus induction, prior to detectable morphological changes. Subsequent changes in gene expression at later regeneration stages were more than an order of magnitude smaller. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression encoded proteins active in auxin and cytokinin signaling, cell division, and plastid development. When compared with data on in vitro callogenesis from root explants in Arabidopsis, 25% (1,260) of up-regulated and 22% (748) of down- regulated genes were in common with the genes that we found regulated in poplar during callus induction. When ~3kb of the 5' flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50 to 60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40% and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3 to 15 days after explants were placed onto callus inducing medium. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Surprisingly, both paralogs of poplar STM were down-regulated 3- to 6-fold during early callus initiation, a possible consequence of its stronger expression in the secondary meristem (cambium) than in shoot tissues. We identified 15 to 35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the WUS and STM transgenic events produced should be useful for monitoring the timing and location of meristem development during natural and in vitro shoot regeneration. / Graduation date: 2008

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bioestimulantes proporcionam incrementos no desenvolvimento vegetal quando aplicados isolados ou em associação com outros produtos. Todavia poucos estudos abordam aspectos vegetativos e produtivos relacionados à aplicação desses em gramíneas forrageiras. Diante disso, o presente trabalho teve como objetivo o estudo das respostas de gramíneas submetidas à aplicação de Bioestimulantes e fertilização foliar. Instalou-se dois experimentos individuais, utilizando a Brachiaria híbrida Convert HD364, na Fazenda Capim Branco, pertencente à Universidade Federal de Uberlândia. O primeiro experimento foi conduzido em delineamento em blocos casualizados, com seis tratamentos e quatro repetições. Os tratamentos foram doses de bioestimulantes (0,25; 0,5; 0,75; 1,00 e 1,25 L ha-1 em cada aplicação) e a testemunha que constou-se da ausência de aplicação do Bioestimulante. O segundo experimento foi conduzido em delineamento inteiramente casualizados, com 7 tratamentos e 3 repetições. Os tratamentos utilizados foram: testemunha (ausência de adubação), aplicação de 30 Kg ha-1 de N (ureia); 30 Kg ha-1 de N (ureia) + 3,0 L ha-1 de Mastermins® Pastagem; 30 Kg ha-1 de N (ureia) + 3,0 L ha-1 de Starter®; 30 Kg ha-1 de N (ureia) + 0,5 L ha-1 de Bioestimulante; 30 Kg ha-1 de N (ureia) + 3,0 L ha-1 de Mastermins® Pastagem + 0,5 L ha-1 de Bioestimulante; 30 Kg ha-1 de N (ureia) + 3,0 L ha-1 de Starter® + 0,5 L ha-1 de Bioestimulante, aplicados em cada ciclo, após o corte. Para os dois experimentos utilizou-se os tratos culturais recomendados para a Brachiaria híbrida Convert. As avaliações realizadas foram: produção de forragem, valor nutritivo (PB, FDN e FDA), massa de raízes e análise foliar para teores de nutrientes. Realizou-se o teste de Regressão para o primeiro experimento e o teste de Tukey a 0,05 de significância para o segundo experimento. Para o primeiro experimento, concluiu que o bioestimulante promove aumento no acúmulo de MS e na taxa de acúmulo de forragem, folhas e colmos de Brachiaria híbrida, redução no percentual de material morto e na relação de F:C Brachiaria híbrida e não interfere nos teores de N, K, P, Ca, Mg, S, B, Cu, Fe, Mn e Zn, o acúmulo de K, P, Ca, Mg, S, B, Cu, Fe, Mn e Zn, o acúmulo de MS e a taxa de acúmulo de material morto, a densidade de raízes, e percentual de folha e as concentrações de FDA, FDN e PB de Brachiaria híbrida Convert HD364, a partir dos resultados encontrados recomenda-se a dose de 1,25 L ha-1 de bioestimulante para Brachiaria híbrida Convert HD364 e para o segundo experimento, concluiu que Starter N® associado ao bioestimulante e ureia promove maior acúmulo de MS de forragem. Starter N® associado a ureia, Mastermins Pastagens® associado a ureia e bioestimulante e Starter N® associado a ureia e ao bioestimulante promovem aumentos no acúmulo de MS de folhas e de colmos. Starter® associado a ureia, Mastermins Pastagens® associado a ureia, Mastermins Pastagens® associado a ureia e ao bioestimulante e Starter® associado a ureia e ao bioestimulante promove aumento no acúmulo de MS de material morto. Mastermins Pastagens® associado a ureia e ao bioestimulante e Starter® associado a ureia e ao bioestimulante promove aumento na taxa de acúmulo de folha. Starter® associado a ureia, Mastermins Pastagens® associado a ureia e ao bioestimulante e Starter® associado a ureia e ao bioestimulante promove aumento taxa de acúmulo de colmo. / Bioestimulants provide increments in plant development when applied alone or in combination with other products. However, few studies address aspects vegetative and productive, related to the application of these in forage grasses. In addition, the objective of this work was to study the responses of grasses, submitted the application of bioestimulants and fertilization. It was installed two experiments, using the Brachiaria hybrid Convert HD364, on the Capim Branco Farm, belonging to the Federal University of Uberlândia. The first experiment was conducted in randomized blocks with six treatments and four replications. The treatments were doses of biostimulation (0.25; 0.5; 0.75; 1.00 and 1.25 L ha-1 in each application) and the witness who appeared to be the lack of application of bioestimulant. The second experiment was conducted in a completely randomized block design, with seven treatments and three replications. The treatments were: control (absence of fertilization), application of 30 kg ha-1 N (urea); 30 kg ha-1 N (urea) + 3.0 L ha-1 of Mastermins® Pasture; 30 kg ha-1 N (urea) + 3.0 L ha-1 of Starter®; 30 kg ha-1 N (urea) + 0.5 L ha-1 bioestimulant; 30 kg ha-1 N (urea) + 3.0 L ha-1 of Mastermins® Pasture + 0.5 L ha-1 bioestimulant; 30 kg ha-1 N (urea) + 3.0 L ha-1 of Starter® + 0.5 L ha-1 of bioestimulant, applied in each cycle, after the cut. The evaluations were forage production, nutritive value (CP, NDF and ADF), and root mass and leaf analysis for nutrient content. There was a regression testing for the first experiment and the Tukey test at 0.05 significance for the second experiment. For the first experiment, concluded that the bioestimulant promotes an increase in the accumulation of MS and the rate of accumulation of grass, leaves and stems of Brachiaria hybrid, a reduction in the percentage of dead material and in relation to F:C Brachiaria hybrid and does not interfere in the contents of N, K, P, Ca, Mg, S, B, Cu, Fe, Mn and Zn, the accumulation of K, P, Ca, Mg, S, B, Cu, Fe, Mn and Zn, the accumulation of MS and the rate of accumulation of dead material, the density of roots, and percentage of leaf and the concentrations of ADF, NDF and PB, from the results it is recommended that the dose of 1.25 L ha-1 bioestimulant for Brachiaria hybrid, Convert HD364. For the second experiment, concluded that Starter N® associated with the bioestimulant and urea promotes greater accumulation of MS of forage. Starter N® associated with urea, Mastermins Pastureland® associated with urea and biostimulating and Starter N® associated with urea and the bioestimulant promote increases in accumulation of MS from leaves and stems. Starter® associated with urea, Mastermins Pastureland® associated with urea, Mastermins Pastureland® associated with urea and the bioestimulant and Starter® associated with urea and the bioestimulant promotes an increase in the accumulation of MS of dead material. Mastermins Pastureland® associated with urea and the bioestimulant and Starter® associated with urea and the bioestimulant promotes an increase in the rate of accumulation of leaf. Starter® associated with urea, Mastermins Pastureland® associated with urea and the bioestimulant and Starter® associated with urea and the bioestimulant promotes increased rate of accumulation of stalk rot. / Dissertação (Mestrado)

Salinity is a challenge for crop productivity. Hence, plants exposed to saline environments reduce their vegetative and reproductive growth due to adverse effects of specific ions on metabolism and water relations. In order to cope with salinity, plants display physiological mechanisms based on three main aspects: i) source-sink relationships, ii) resource allocation and iii) alterations in endogenous hormone levels. The roles of developmental and hormonal mechanisms in salt response were investigated here. We employed mutants and transgenic tomato plants affecting different aspects of plant development and hormone response in the same genetic background (cultivar Micro-Tom). The following genotypes were used: Galapagos dwarf (Gdw), Lanata (Ln), lutescent (l), single flower truss (sft), sft heterozygous (sft/+), diageotropica (dgt), entire (e), Never ripe (Nr), epinastic (epi), procera (pro), notabilis (not), anti sense Chloroplastic carotenoid cleavage dioxygenase 7 (35S::asCCD7) and Salicylate hydroxylase (35S::nahG). Among the developmental genotypes studied, sft and l, involved in flower induction and senescence, respectively, were less affected when exposed to salt stress. Although l is considered deleterious due to its precocious senescence, it presented greater shoot biomass and leaf area during salinity. The heterozygous sft/+, whose high productivity was recently linked to an improved vegetative-to-reproductive balance, changed this balance and lowered its yield more than the control MT upon salt treatment. In the analysis of genotypes affecting hormonal status/signaling four kinds of salt responses among the genotypes were observed: i) High shoot growth in spite of high Na:K ratio presented by the strigolactone deficient and high branching CCD7 transgene; ii) High shoot growth and reduced accumulation of Na in tissues (probably due to dilution) presented by the auxin constitutive response e mutant; iii) The opposite response observed in \"ii\" presented by the low auxin sensitivity dgt mutant and iv) growth inhibition combined with reduced levels of Na and higher accumulation of K presented by the not mutant, which produces less ABA. Taken together, the results presented here points to novel developmental mechanisms, such as the promotion of moderate senescence and vegetative growth, and hormonal imbalances to be explored in the pursuing of crops resistant to salt stress. / A salinidade é um desafio para a produtividade agrícola, uma vez que plantas expostas à salinidade tem o crescimento vegetativo e reprodutivo reduzido devido aos efeitos adversos de íons específicos no metabolismo e nas relações hídricas. A fim de lidar com a salinidade, as plantas desempenham mecanismos fisiológicos baseados em três principais características: i) relações fonte-dreno; ii) alocação de reservas e iii) alterações nos níveis endógenos de hormônios. Nesse trabalho, investigamos a relação entre os processos de desenvolvimento e de regulação hormonal com a resposta à salinidade. Para tanto foram usados genótipos de tomateiro com alteração em diferentes vias de desenvolvimento e de produção ou sinalização de hormônios vegetais. Os seguintes genótipos foram usados: Galapagos dwarf (Gdw), Lanata (Ln), lutescent (l), single flower truss (sft), sft heterozygous (sft/+), diageotropica (dgt), entire (e), Never ripe (Nr), epinastic (epi), procera (pro), notabilis (not), anti sense Dioxigenase cloroplastídica de carotenoide 7 (35S::asCCD7) e Salicilato hidroxilase (35S::nahG). Entre os genótipos de desenvolvimento estudados, sft e l, relacionados à menor indução floral e senescência respectivamente, foram os menos afetados quando expostos à salinidade. O genótipo l acumulou maior biomassa e área foliar, apesar de ser considerado deletério devido à senescência precoce. As plantas heterozigotas, sft/+, cuja maior produtividade foi recentemente relacionada a um melhor balanço vegetativo/reprodutivo, alteraram esse balanço sob salinidade e reduziram sua produtividade mais que o controle MT sob estresse salino. Na análise dos genótipos com alteração hormonais foram observados quatro tipos de respostas à salinidade: i) elevado crescimento da parte aérea, apesar da razão Na:K ser alta no genótipo CCD7 cujo transgene induz deficiência de estrigolactona e excessiva ramificação; ii) elevado crescimento e acúmulo reduzido de Na nos tecidos (devido provavelmente a diluição) apresentada pelo mutante de resposta constitutiva a auxina e; iii) o oposto da resposta anterior foi apresentado pelo mutante pouco sensível à auxina , dgt; iv) inibição do crescimento combinado com nível reduzido de Na e alto acúmulo de K apresentada pelo mutante not que produz menos ácido abscísico. Considerados em conjunto, os resultados apresentaram temas para novos mecanismos de desenvolvimento, como a promoção moderada de senescência e do crescimento vegetativo além dos desbalanços hormonais, para serem explorados na busca de culturas resistentes ao estresse salino.

Salinity is a challenge for crop productivity. Hence, plants exposed to saline environments reduce their vegetative and reproductive growth due to adverse effects of specific ions on metabolism and water relations. In order to cope with salinity, plants display physiological mechanisms based on three main aspects: i) source-sink relationships, ii) resource allocation and iii) alterations in endogenous hormone levels. The roles of developmental and hormonal mechanisms in salt response were investigated here. We employed mutants and transgenic tomato plants affecting different aspects of plant development and hormone response in the same genetic background (cultivar Micro-Tom). The following genotypes were used: Galapagos dwarf (Gdw), Lanata (Ln), lutescent (l), single flower truss (sft), sft heterozygous (sft/+), diageotropica (dgt), entire (e), Never ripe (Nr), epinastic (epi), procera (pro), notabilis (not), anti sense Chloroplastic carotenoid cleavage dioxygenase 7 (35S::asCCD7) and Salicylate hydroxylase (35S::nahG). Among the developmental genotypes studied, sft and l, involved in flower induction and senescence, respectively, were less affected when exposed to salt stress. Although l is considered deleterious due to its precocious senescence, it presented greater shoot biomass and leaf area during salinity. The heterozygous sft/+, whose high productivity was recently linked to an improved vegetative-to-reproductive balance, changed this balance and lowered its yield more than the control MT upon salt treatment. In the analysis of genotypes affecting hormonal status/signaling four kinds of salt responses among the genotypes were observed: i) High shoot growth in spite of high Na:K ratio presented by the strigolactone deficient and high branching CCD7 transgene; ii) High shoot growth and reduced accumulation of Na in tissues (probably due to dilution) presented by the auxin constitutive response e mutant; iii) The opposite response observed in \"ii\" presented by the low auxin sensitivity dgt mutant and iv) growth inhibition combined with reduced levels of Na and higher accumulation of K presented by the not mutant, which produces less ABA. Taken together, the results presented here points to novel developmental mechanisms, such as the promotion of moderate senescence and vegetative growth, and hormonal imbalances to be explored in the pursuing of crops resistant to salt stress. / A salinidade é um desafio para a produtividade agrícola, uma vez que plantas expostas à salinidade tem o crescimento vegetativo e reprodutivo reduzido devido aos efeitos adversos de íons específicos no metabolismo e nas relações hídricas. A fim de lidar com a salinidade, as plantas desempenham mecanismos fisiológicos baseados em três principais características: i) relações fonte-dreno; ii) alocação de reservas e iii) alterações nos níveis endógenos de hormônios. Nesse trabalho, investigamos a relação entre os processos de desenvolvimento e de regulação hormonal com a resposta à salinidade. Para tanto foram usados genótipos de tomateiro com alteração em diferentes vias de desenvolvimento e de produção ou sinalização de hormônios vegetais. Os seguintes genótipos foram usados: Galapagos dwarf (Gdw), Lanata (Ln), lutescent (l), single flower truss (sft), sft heterozygous (sft/+), diageotropica (dgt), entire (e), Never ripe (Nr), epinastic (epi), procera (pro), notabilis (not), anti sense Dioxigenase cloroplastídica de carotenoide 7 (35S::asCCD7) e Salicilato hidroxilase (35S::nahG). Entre os genótipos de desenvolvimento estudados, sft e l, relacionados à menor indução floral e senescência respectivamente, foram os menos afetados quando expostos à salinidade. O genótipo l acumulou maior biomassa e área foliar, apesar de ser considerado deletério devido à senescência precoce. As plantas heterozigotas, sft/+, cuja maior produtividade foi recentemente relacionada a um melhor balanço vegetativo/reprodutivo, alteraram esse balanço sob salinidade e reduziram sua produtividade mais que o controle MT sob estresse salino. Na análise dos genótipos com alteração hormonais foram observados quatro tipos de respostas à salinidade: i) elevado crescimento da parte aérea, apesar da razão Na:K ser alta no genótipo CCD7 cujo transgene induz deficiência de estrigolactona e excessiva ramificação; ii) elevado crescimento e acúmulo reduzido de Na nos tecidos (devido provavelmente a diluição) apresentada pelo mutante de resposta constitutiva a auxina e; iii) o oposto da resposta anterior foi apresentado pelo mutante pouco sensível à auxina , dgt; iv) inibição do crescimento combinado com nível reduzido de Na e alto acúmulo de K apresentada pelo mutante not que produz menos ácido abscísico. Considerados em conjunto, os resultados apresentaram temas para novos mecanismos de desenvolvimento, como a promoção moderada de senescência e do crescimento vegetativo além dos desbalanços hormonais, para serem explorados na busca de culturas resistentes ao estresse salino.

Catharanthus roseus est une plante tropicale qui produit spécifiquement des alcaloïdes indoliques monoterpéniques (AIM) d’intérêt thérapeutique. Chez C. roseus, la branche terpénique incluant la voie du méthylérythritol phosphate (MEP) est considérée comme limitante et présente une régulation transcriptionnelle coordonnée en réponse aux hormones inductrices de l’accumulation alcaloïdique. Lors de ce travail, suite à des analyses bioinformatiques et à la caractérisation de promoteurs de gènes de la voie MEP, nous avons identifié de nouvelles familles de facteurs de transcription impliquées dans la régulation de la biosynthèse des AIM. Des membres de la famille des ZCT, des WRKY et des RR type B interagissent avec le promoteur du gène hds de la voie MEP et régulent son activité. Ces travaux ont permis d’approfondir les connaissances sur les réseaux transcriptionnels régulateurs de la biosynthèse des AIM. L’utilisation de ces nouveaux facteurs de transcription activateurs peut désormais être envisagée dans le cadre d’expériences d’ingénierie métabolique afin d’augmenter l’accumulation d’alcaloïdes d’intérêt pharmaceutique chez C. roseus. / Catharanthus roseus is a tropical plant producing specifically monoterpene indole alkaloids (MIA) of high interest due to their therapeutical values. In C. roseus cells, the terpenoid branch including the methyl erythritol phosphate pathway (MEP) provides the MIA terpenoid moiety and is regarded as limited for MIA biosynthesis. This branch presents a coordinated transcriptional regulation in response to hormonal signals leading to MIA production. In this context, bioinformatic analysises and functional characterization of MEP pathway gene promoters allowed the identification of new transcription factor families involved in the MIA pathway regulation. Members of ZCT proteins, WRKY and type B RR families specifically interact with the hds promoter from the MEP pathway and regulate its activity. This work permits to gain into insight the transcriptional network controlling the MIA biosynthesis. It is possible now to consider using transcription factor that act as activators and target genes from the terpenoid branch to increase the accumulation of alkaloids of pharmaceutical interest in C. roseus by metabolic engineering approaches.