As proteínas-cinases ativadas por mitógenos (MAPKs) participam de rotas de transdução de sinais universais em eucariotos e compõem cascatas de fosforilação que levam as células a responder a estímulos extracelulares. Em plantas, MAPKs estão associadas a processos de desenvolvimento e na reposta a hormônios e a estresses bióticos e abióticos. Em trabalhos anteriores de nosso grupo, o gene Egmpk5 de Eucalyptus grandis foi clonado e seu padrão de expressão foi caracterizado em plantas submetidas a diferentes tratamentos. Ainda, plantas de Nicotiana tabacum SR1 foram transformadas com Egmpk5 sob regulação do promotor CaMV 35S para futuro estudo de função do gene. No presente trabalho, foi avaliada mais amplamente a estrutura, a expressão e o produto deste gene. Análises in silico revelaram que Egmpk5 apresenta-se como cópia única no genoma, está organizado em seis éxons e seus cinco íntrons possuem sítios canônicos de splicing de RNA. O produto deduzido deste gene apresenta em sua sequência a assinatura do domínio proteína-cinase e os motivos de ligação a ATP e de ativação/dupla fosforilação, indicando a possibilidade de ser uma proteína funcional. Árvore filogenética foi construída pelo método de Máxima Verossimilhança utilizando o algoritmo MUSCLE para alinhamento das sequências e um valor de bootstrap de 500 repetições com sequências peptídicas similares à proteína deduzida EgMPK5 e permitiu a identificação de MAPKs de outras plantas com função já comprovada na sinalização da divisão celular e na resposta a estresses abióticos, ferimento e patógenos. Análise de RT-qPCR mostrou a expressão de Egmpk5 em níveis comparáveis em raízes, caules e folhas de plantas de E. grandis tratadas com água, e foi detectado um aumento de sua expressão em folhas tratadas com ácido abscísico (ABA) e em todos os órgãos tratados com solução de cloreto de sódio a 200 mM. O estudo da região promotora de Egmpk5 revelou um grupo de possíveis elementos de ação cis relacionados a respostas a estresses. Seis linhagens transformadas de tabaco que tiveram seu estado transgênico confirmado por PCR foram desafiadas com tratamentos de seca e cloreto de sódio, e linhagens mais tolerantes foram identificadas. Ao final destas diversas análises, os resultados obtidos sugerem a participação de Egmpk5 em respostas a estresses abióticos. / Mitogen-activated protein kinases (MAPKs) are part of phosphorylation cascades found in all eukaryotes and are responsible for transducing extracellular stimuli into intracellular responses. In plants, MAPK cascades are involved in growth and development processes and have roles in the response to hormones and biotic and abiotic stresses. In our previous studies, the Egmpk5 gene from Eucalyptus grandis was cloned and its expression profile was characterized in plants subjected to different treatments. Also, Nicotiana tabacum SR1 plants were transformed with Egmpk5 under the control of the CaMV 35S promoter for further function studies. In the present study the structure, expression profiles and product of this gene were further characterized. In silico analyses revealed that the Egmpk5 gene is present as a single copy in the E. grandis genome, and that its structure comprises six exons and five introns with conserved sites for transcript splicing. The deduced product of this gene presents the protein kinase domain signature, ATP-binding site and activation/dual phosphorylation motifs in its structure. A phylogenetic tree was built with sequences similar to the deduced EgMPK5 peptide and lead to the identification of plant MAPKs that have had proven activity in cell division signaling and in the response to abiotic stresses, wounding and pathogens. RT-qPCR analysis showed equal expression of Egmpk5 in roots, stems and leaves of plants treated with water and we detected an increase of expression in leaves treated with abscisic acid (ABA) and also increase in expression in all three organs upon treatment with 200 mM sodium chloride. Analysis of the promoter region of Egmpk5 revealed a group of putative cis-acting elements related to stress responses. Six transformed tobacco lines were confirmed by PCR and were assayed with drought and sodium chloride treatments for the identification of more tolerant individuals. After all analyses performed, results allowed us to suggest that Egmpk5 is involved in abiotic stresses responses.

Orientador: Lourival Carmo Monaco / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-17T17:52:59Z (GMT). No. of bitstreams: 1 Pires_CesarioLangedaSilva_M.pdf: 4614774 bytes, checksum: dc31db7ca2543960b24d9bbaf5aa8bb5 (MD5) Previous issue date: 1983 / Resumo: Alfa esterase e beta esterase foram estudadas em plântulas no estádio "orelha de onça" de 20 populações de E.grandis (19 australianas e 1 brasileira) e 2 de E. saligna (1 australiana e 1 brasileira) em gel de amido. Foi testado o estudo de 11 sistemas enzimáticos para a determinação das diferenças genéticas dos 22 tratamentos. Inicialmente foram escolhidos como os de melhor resolução a leucina aminopeptidase, alfa esterase, beta esterase e fosfatase ácida. Porém pela perfeição da resolução obtida, só foram estudadas as frequências de 3 possíveis locos de alfa esterase e 3 de beta esterase, os quais forneceram informações suficientes apenas para a identificação da população G1l, da S1 e de um grupo formado por todas as outras populações. As diferenças pequenas, entre as populações sugerem uma apreciação recente. Os dados sugerem que a população brasileira de E. Saligna se afastou mais da espécie tipo do que E. grandis (de sua espécie tipo). Uma das possíveis causas seria a maior facilidade de cruzamento dentro do subgênero. O isolamento das populações do planalto de Atherton determinou distâncias genéticas entre si maiores do que a distância entre as populações do planalto e as do sul. As distâncias entre as populações estudadas e situadas ao sul do planalto, foram bem menores. Das populações do planalto destaca-se a de E de Mareeba com frequência de alelos bem distinta. ...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Alfa esterase and beta esterase were studied in seedlings (before the emergency of the first nomophyllous leaves and possessing on1y the pair of cotyledonary 1eaves) of 20 E. grandis populations (19 from Australia and 1 from Brazil) and 2 E. saligna (1 from Australia and 1 from Brazil), using the horizontal electrophoresis technique in Connaught starch gel. Eleven enzymatic systems were tested to determine the genetic differences among the 22 populations. Leucine aminopeptidase, alfa esterase, beta esterase and acid phosphatase had reasonable good resolution. Because the perfection of the resolution, on1y the frequency of 3 possible alfa esterase loci and 3 possible beta esterase loci were studied which gave not sufficient information to permit a proper identification of different populations. It was possib1e to identify the population G11, from the S1 and from a group formed by the other populations. The small differences among the populations suggest a recent speciation. The data suggest that the Brasilian population of E. saligna is genetically more distant from the tipical species than E. grandis from its species. The possible reason is that E. saligna cross more easily with the other species of the subgenus. The isolation of the populations at the Atherton table-land determined genetic distances among them greater than the distance between the Atherton populations and the southern ones. The distance among the southern populations were smaller. The population of Mareeba presented a distinct allele frequency. ...Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Mestre em Ciências Biológicas

Orientador: Mario Luiz Teixeira de Moraes / Coorientador: Edson Seizo Mori / Banca: Alexandre Magno Sebbenn / Banca: Leo Zimback / Resumo: O Eucalyptus grandis é uma das espécies mais plantadas no Brasil devido às características silviculturais e a sua adaptação a diferentes condições edafoclimáticas, o que possibilita resultados significativos de rendimento volumétrico. Diversas procedências e progênies vêm sendo estudadas com o propósito de se conhecer a estrutura genética destas populações, o que facilitará a obtenção de material genético adequado ao desenvolvimento de programas de melhoramento florestal no país. O objetivo deste trabalho foi realizar o zoneamento ecológico por meio da análise de estabilidade, adaptabilidade e produtividade de progênies de Eucalyptus grandis, utilizando-se do método MHPRVG (média harmônica da performance relativa dos valores genéticos) preditos por BLUP e estimar a variabilidade genética a partir de caracteres quantitativos. Foram instalados quatro testes de progênies de polinização aberta nos municípios de Anhembi, Avaré, Itararé e Pratânia. O delineamento experimental utilizado foi o de blocos casualizados, com quatro e seis repetições e seis plantas por parcela. Um total de 153 a 160 tratamentos (progênies) foram avaliados para diâmetro a altura do peito, altura das árvores e volume, nos quatro ensaios as medições foram realizadas aos 12 e 24 meses de idade. Os parâmetros genéticos, para o estudo de estabilidade, adaptabilidade e produtividade dos genótipos de E. grandis, foram estimados por meio da metodologia REML/BLUP. Os altos valores encontrados para o coeficiente de variação genética demostram que existe alta variabilidade genética entre as progênies, corroboradas com altas herdabilidades médias, os efeitos da interação genótipos ambientes foram significativos. O método da MHPRVG permitiu a seleção de progênies com alto potencial produtivo predito, classificadas simultaneamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Eucalyptus grandis is a species grown in Brazil due to forest features and their adaptation to different climatic conditions, which enables significant results of volumetric efficiency. Several provenances and progenies have been studied in order to understand the genetic structure of these populations, which will facilitate the acquisition of genetic material suitable for development of forest breeding programs in the country. The objective of this study was realize the ecological zoning through the analysis of stability, adaptability and productivity of progenies of Eucalyptus grandis, using the method MHPRVG (harmonic mean of the relative performance of the predicted genetic values) predicted by BLUP and estimate the genetic variability from quantitative traits. Were installed four sites of Open-pollinated progenies in the municipalities of Anhembi, Avaré, Itararé and Pratânia. Were established in a randomized complete blocks design with the families at each site numbering from 153 to 160, the blocks at each site numbering from four to six and the trees per plot ranging from to six, were evaluated for trees diameter at breast height, height and volume, at four sites measurements were at 12 and 24 months. The genetics parameters for study of stability, adaptability and productivity of genotype of E. grandis, were estimate by genetic REML/BLUP. The high values found for coefficient of genetic variation showing high genetic variability among progenies, corroborated with high heritability means, the effects of genotype x environments were significant. The method of MHPRVG allowed the selection of progenies with high yield potential predicted, classified simultaneous by productivity, stability and adaptability. The high values of heritability means of progenies above 67% progeny allow anticipating the successful selection of... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:30:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-19Bitstream added on 2014-06-13T20:40:15Z : No. of bitstreams: 1 miranda_ac_me_botfca.pdf: 352756 bytes, checksum: e94f547138bf0223054b6f08ecb6ee76 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O Eucalyptus grandis é uma das espécies mais plantadas no Brasil devido às características silviculturais e a sua adaptação a diferentes condições edafoclimáticas, o que possibilita resultados significativos de rendimento volumétrico. Diversas procedências e progênies vêm sendo estudadas com o propósito de se conhecer a estrutura genética destas populações, o que facilitará a obtenção de material genético adequado ao desenvolvimento de programas de melhoramento florestal no país. O objetivo deste trabalho foi realizar o zoneamento ecológico por meio da análise de estabilidade, adaptabilidade e produtividade de progênies de Eucalyptus grandis, utilizando-se do método MHPRVG (média harmônica da performance relativa dos valores genéticos) preditos por BLUP e estimar a variabilidade genética a partir de caracteres quantitativos. Foram instalados quatro testes de progênies de polinização aberta nos municípios de Anhembi, Avaré, Itararé e Pratânia. O delineamento experimental utilizado foi o de blocos casualizados, com quatro e seis repetições e seis plantas por parcela. Um total de 153 a 160 tratamentos (progênies) foram avaliados para diâmetro a altura do peito, altura das árvores e volume, nos quatro ensaios as medições foram realizadas aos 12 e 24 meses de idade. Os parâmetros genéticos, para o estudo de estabilidade, adaptabilidade e produtividade dos genótipos de E. grandis, foram estimados por meio da metodologia REML/BLUP. Os altos valores encontrados para o coeficiente de variação genética demostram que existe alta variabilidade genética entre as progênies, corroboradas com altas herdabilidades médias, os efeitos da interação genótipos ambientes foram significativos. O método da MHPRVG permitiu a seleção de progênies com alto potencial produtivo predito, classificadas simultaneamente... / The Eucalyptus grandis is a species grown in Brazil due to forest features and their adaptation to different climatic conditions, which enables significant results of volumetric efficiency. Several provenances and progenies have been studied in order to understand the genetic structure of these populations, which will facilitate the acquisition of genetic material suitable for development of forest breeding programs in the country. The objective of this study was realize the ecological zoning through the analysis of stability, adaptability and productivity of progenies of Eucalyptus grandis, using the method MHPRVG (harmonic mean of the relative performance of the predicted genetic values) predicted by BLUP and estimate the genetic variability from quantitative traits. Were installed four sites of Open-pollinated progenies in the municipalities of Anhembi, Avaré, Itararé and Pratânia. Were established in a randomized complete blocks design with the families at each site numbering from 153 to 160, the blocks at each site numbering from four to six and the trees per plot ranging from to six, were evaluated for trees diameter at breast height, height and volume, at four sites measurements were at 12 and 24 months. The genetics parameters for study of stability, adaptability and productivity of genotype of E. grandis, were estimate by genetic REML/BLUP. The high values found for coefficient of genetic variation showing high genetic variability among progenies, corroborated with high heritability means, the effects of genotype x environments were significant. The method of MHPRVG allowed the selection of progenies with high yield potential predicted, classified simultaneous by productivity, stability and adaptability. The high values of heritability means of progenies above 67% progeny allow anticipating the successful selection of... (Complete abstract click electronic access below)

Orientador: Giuseppina Pace Pereira Lima / Coorientador: Elizabeth Orika Ono / Banca: Luciana Francisco Freire / Banca: Armando Reis Tavares / Resumo: A propagação vegetativa é a principal forma de produção de mudas de Eucalyptus, porém alguns materiais clonais ainda apresentam dificuldades de propagação, inclusive no enraizamento. Indiretamente, os flavonóides e o peróxido de hidrogênio podem favorecer este processo, assim como alterar os níveis de substâncias antioxidantes, como enzimas e compostos fenólicos. O objetivo deste trabalho foi avaliar a ação do flavonóide quercetina, peróxido de hidrogênio e ácido indol butírico (IBA) no enraizamento e desenvolvimento de Eucalyptus grandis x urophylla, bem como nos mecanismos de produção de antioxidantes e proteção das células contra danos oxidativos. O estudo foi dividido em duas fases. Inicialmente os clones foram submetidos a tratamentos com combinação de peróxido de hidrogênio (0; 1,75; 3,5 e 7,0%) e IBA (0; 1.000 mg kg-1) e a combinação de quercetina (0; 250; 500; 1.000 mg kg-1) e IBA (0; 1.000 mg kg-1). Os tratamentos foram aplicados em estacas e as mudas foram avaliadas em relação aos trinta, sessenta e noventa dias após o plantio em relação a altura, diâmetro e porcentagem de sobrevivência. Aos noventa dias uma avaliação destrutiva foi realizada e determinados a porcentagem de enraizamento, o tamanho médio da maior raiz e a quantidade de raízes. Após esta avaliação, o peróxido de hidrogênio (0;3,5%), IBA (0; 1.000 mg kg-1) e a quercetina (0; 500 mg kg-1) foram combinados a fim de verificar a interação de peróxido de hidrogênio, quercetina e IBA sobre algumas moléculas com capacidade antioxidante. Noventa dias após o plantio, os efeitos desses compostos na atividade das enzimas IAA-oxidase, peroxidase, polifenoloxidase, fenilalanina amônio liase e poliamina oxidase e nos teores de compostos fenólicos, flavonoides, antocianinas e poliaminas foram avaliados por métodos espectofotométricos e teores de quercetina e ácido indolacético foram avaliados ... / Abstract: Vegetative propagation is the main form to produce cuttings of Eucalyptus, but some clones still have difficulties in propagation, including on rooting process. Indirectly, flavonoids and hydrogen peroxide can enhance this process, as well as modify the levels of antioxidants such as phenolic compounds and enzymes. This work was aimed to evaluate flavonoid quercetin, hydrogen peroxide and indolbutyric acid (IBA) action on rooting of cuttings of Eucalyptus grandis x urophylla, as well as in mechanisms of antioxidants production and cells protection against oxidative damage. The study was divided into two phases. Initially, clones were subjected to treatments with combination of hydrogen peroxide (0, 1.75, 3.5 and 7.0%) and IBA (0, 1.000 mg kg-1) and combination of quercetin (0, 250, 500, 1.000 mg kg-1) and IBA (0, 1.000 mg kg-1). Treatments were applied to cuttings and evaluated in thirty, sixty and ninety days after planting in relation to height, diameter and survival percentage. Ninety days after planting, a destructive evaluation was performed and percentage of rooting, average size of the largest root and the amount of roots were determined. After this evaluation, hydrogen peroxide (0, 3.5%), IBA (0, 1000 mg kg-1) and quercetin (0,500 mg kg-1) were combined in order to verify the interaction of hydrogen peroxide, quercetin and IBA on some molecules with antioxidant capacity. Ninety days after planting, these compounds effects on the activity of IAA oxidase, peroxidase, polyphenol oxidase, phenylalanine ammonia lyase and polyamine oxidase and the levels of phenolics, flavonoids, anthocyanins and polyamines were evaluated by spectrophotometric methods and levels of quercetin and indole acetic acid were evaluated via LC/MS in leaves and roots. Data were subjected to analysis of variance (ANOVA) and Tukey test at 5 % probability. No significant differences were found in relation to height, diameter, survival ... / Mestre

Eucalyptus grandis and its hybrids is the most important and widely planted eucalypt in South Africa. It has a wide range of uses including pulpwood, poles, firewood, charcoal, flooring, mining, furniture and general carpentry. Conservation of plant genetic resources including those used in agriculture, horticulture and forestry has become an issue of common global concern. Cryopreservation involves the storage of plant material at ultra low temperature (-196°C). The techniques for cryopreservation currently in use are varied and include the older classical techniques and the new vitrification-based techniques. Storage of biological material at -196°C causes metabolic functions to slow down considerably and minimize biological degradation, thus allowing for long-term preservation. However, there are particular stresses associated with the freezing process, e.g., ice crystal formation and cryo-dehydration, which may severely damage the material. Tolerance to drying is the key to successful cryopreservation and is commonly used in the preparation of in vitro material for cryostorage. However, drying may result in damages and a number of stresses that may activate caspase-like proteases and trigger cell death processes such as programmed cell death and necrosis. During the drying process, the physical and physiological characteristic of the cell changes because of the removal of water and damage is reflected by the lack of resumption of normal activity upon rehydration. As part of a cryo-procedure, Eucalyptus grandis axillary buds isolated from in vitro shoots were dried over silica gel for 20 minutes. Pre-treatment of the shoots with 5mg.l-1 ABA for 5 days resulted in partial resistance of the isolated buds to water loss (76% to 45%) as compared with untreated buds (76% to 33%). Concomitantly, viability decreased from 100 to 70% for ABA treated buds and to 55% for the untreated buds. Ultrastructural examination showed cellular responses to drying, ranging from cell death, through partial disruption to organelles to apparently normal ultrastructure. The use of the vital stains, 4,6-diamidino-2-phenylindole and propidium iodide, showed that certain regions of the buds (e.g. the leaf primordia) were the most prone to drying damage. The meristem, however, appeared to survive drying and for up to 72 hours of rehydration. High Reactive Oxygen Species (ROS) activity was associated with bud excision and the drying procedure. Caspase-3-like protease activity was detected after drying and rehydration in both nonviable treated and untreated buds, but not in the hydrated controls. The Caspase-3 inhibitors Ac-DEVD-CHO, pepstatin and leupeptin partially suppressed that activity. The ultrastructural studies and the use of the vital stains provided confirmation of the beneficial effects of ABA. The detection of a caspase-3- like protease has provided some evidence that the rehydrated buds, that had ultimately died, had undergone programmed cell death. The ROS production during bud isolation which was exacerbated by the drying procedure is considered to be the trigger for the programmed cell death. Data in the present study showed the role of both necrosis and PCD in the death of the tissues of the axillary buds of E. grandis axillary buds. The data also contributed to the better understanding of the impact of cryoprocedures on these clonal tissues which are ideal propagules for forestry germplasm conservation.

Alien invasive species can have serious negative impacts on the biodiversity and functioning of ecosystems, but identifying invaders early, before they cause problems, can dramatically reduce the costs of controlling them. There is substantial research in identifying key attributes of invasive species, which can potentially be used in this regard. Many Eucalypts have formed the backbone of forestry in South Africa since the 1800s. While many other plantation species such as pines and legumes, have become serious invaders in many parts of the world, Eucalyptus species have been not been nearly as successful in invading alien environments. This is surprising considering that in their native habitat; members of this genus dominate almost all vegetation types. This project used available theory on the qualities that characterise invasive species to assess the invasive potential of one Eucalyptus species: Eucalyptus grandis (rose gum). Many alien plants take a long time to establish naturalised populations and spread through new ecosystems and this research will provide information on the likelihood that E. grandis will become a problem species in the future. A field study was used to determine whether there is any indication that it is in fact, invading from plantations in Mpumalanga, and if so, which ecological processes affect this invasion potential. Belt transects (5 by 50 metres) were used in sampling the populations growing near plantations. To determine whether frost is affecting the populations, one site was at high elevation where it is exposed to frost (near Graskop) and the other at a low elevation area with infrequent frost (near White river). Key reproductive traits such as generation time and seed viability which are known to affect invasion potential were also studied. Demographic data was used to determine the rates of establishment of E. grandis outside of plantations. The results showed that E. grandis had a short generation time and its seeds had a viability of 97%. Assessing the shape parameter (c) of the Weibull distribution function showed that both the Graskop (c=1) and White River (c=1) size class distributions had reverse j-shaped curves, characteristic of good rejuvenation. However, some Graskop sites had a monotonic function (c < 1) showing that frost is affecting the rejuvenation process. Generally the results show that rate of spread is low and this might suggest that the populations are on the establishing populations’ invasion stage. However, there is no indication that there are any environmental or life history factors that would prevent Eucalyptus from becoming invasive in the future, and I would recommend strict monitoring of its rates of spread out of plantation forests in various parts of the country.

Os agrupamentos de ferro-enxofre (Fe-S) são grupos prostéticos necessários para a manutenção da vida, pois estão envolvidos em diversos processos incluindo a transferência de elétrons, reações metabólicas, sinalização e regulação da expressão gênica. As plantas realizam fotossíntese e respiração, dois processos que requerem proteínas Fe-S, sendo os únicos organismos em que a síntese destas proteínas é compartimentalizada. Diversos fatores afetam o desenvolvimento das plantas, entre eles, a temperatura baixa, fator limitante à produtividade e à distribuição geográfica das plantas, incluindo Eucalyptus grandis, uma espécie com grande importância econômica. Neste trabalho foi realizada uma análise transcricional dos genes NFS1, ISA1 e ISU1 de E. grandis após diferentes estúmlos por meio de PCR quantitativa (qRT-PCR) e microarranjos. Após o tratamento de plântulas de E. grandis com frio, foram realizados experimentos de qRT-PCR. Os resultados foram normalizados com os genes constitutivos codificadores da histona H2B e da ribonucleoproteína L23A. Considerando tal normalização, ISU1 aumentou sua expressão em 0,6 e 1,7 vezes, NFS1 apresentou um aumento de 6 e 8 vezes, enquanto ISA1 apresentou um aumento de 69 a 114 vezes em relação à condição controle. Utilizando-se a técnica de microarranjos, foi analisada a diferença de expressão entre folhas e xilema de árvores maduras de E. grandis. O gene NFS1 apresentou maior expressão nas folhas do que em xilema, porém os genes ISA1 e ISU1 apresentaram um padrão de expressão equivalente entre os dois tipos de tecidos. Esses resultados sugerem que (i) os genes NFS1 e ISA1 podem estar relacionados à resposta celular ao estresse causado por frio; e que (ii) os aumentos na expressão devem-se, provavelmente, ao metabolismo de enxofre e à indução de enzimas antioxidantes. Foi também realizado um experimento de curva de tempo com a submissão de plântulas de E. grandis ao resfriamento, objetivando-se verificar em que momento esses genes começam a ter suas expressões aumentadas. O gene ISU1 apresentou maior expressão gênica nas primeiras duas horas de tratamento, caindo drasticamente logo após este período. O gene ISA1, que havia apresentado a maior expressão relativa no experimento anterior, não apresentou diferença significativa no padrão de expressão durante as 16 horas de resfriamento, assim como o gene NFS1. Esses resultados indicam que as proteínas Fe-S, frente ao resfriamento, estão possivelmente envolvidas na recuperação das plantas após tal estresse. / Iron-sulfur (Fe-S) clusters are prosthetic groups required for the maintenance of life because they are involved in various vital processes, including electron transfers, metabolic reactions, signaling and regulation of gene expression. Plants perform photosynthesis and respiration, two processes that require Fe-S proteins, and are the only organisms in which the synthesis of these proteins is compartmentalized. Several factors and stresses affect the development of plants including low temperature, which is a productivity-limiting factor and restricts plants to certain geographical distributions, including Eucalyptus grandis, a species with significant economic importance. The aim of this study is to perform an analysis of E. grandis NFS1 and ISA1 gene expressions after different stimuli through quantitative PCR (qRT-PCR) and microarrays. qRT-PCR experiment were conducted on plants submitted to a cold treatment. The results were normalized with the housekeeping genes encoding histone H2B and ribonucleoprotein L23A. Considering such normalizations, ISU1 increased the expression 0.6 and 1-fold, NFS1 showed a 6 and 8-fold increase in comparison with the control condition, while ISA1 gene increased 69 and 114-fold. Using microarrays, the difference in expression between leaves and xylem of E. grandis was analyzed. The NFS1 gene showed higher expression in leaves than in xylem, but the ISA1and ISU1 showed equivalent pattern of expression in both types of tissues. These results suggest that (i) NFS1 and ISA1 genes are related to the cellular response to the stress caused by chilling, and that (ii) the increased expression should be probably due to the metabolism of sulfur and to the induction of antioxidative enzymes. A time-course experiment was also conducted during the cold stress of E. grandis plants to look at which moment these genes begin to increase their expressions. The ISU1 gene showed higher expression in the first 2 hours of treatment, and than decreased severally after this period. The ISA1 gene, which had shown the highest expression in the previous experiment, did not show significant differences in the pattern of expression during the 16 hours of chilling treatment, as well as the NFS1 gene. These results indicate that Fe-S proteins, in response to low temperature, are possibly involved in the recovery of the plants after this stress.

Hybrid clones of Eucalyptus grandis and E. nitens (GN) have consistently been shown to be suitable for planting in cold, dry, marginal plantation sites, where they exhibit high yields and superior pulp properties. However, their clonal propagation is hindered by the very poor rooting success of cuttings. The present study aimed at assessing the effect of cutting type, time of year of setting cuttings and Seradix 2 application on rooting and development of cuttings of a commercially important Eucalyptus grandis x Eucalyptus nitens clone (GN107). Cuttings were prepared from clonal hedge coppice at the Mondi Business Paper, Trahar Technology Centre, Hilton. Three cutting types were used (cut at different distances from the node) for each terminal (situated below the apical bud) and non-terminal cuttings. The leaves were trimmed and, for half the cuttings, the base of the stem of cuttings were dipped in Seradix 2 rooting powder (3 g kg-1 4-(indole-3-yl)-butyric acid (IBA). They were then placed into rooting trays (128 inserts/ tray arranged as 8 rows x 16 columns). Seradix 2-treated and Seradix 2-untreated terminal and non-terminal cuttings, cut at, above and below the node (twelve treatments in total) were set in trays with one treatment per column of eight replicates, per tray. There were nineteen trays overall. The trays were filled with peat, perlite and vermiculite (3:3:1) and were maintained in a Mondi greenhouse, with air temperature at 25°C to 27°C (thermostatically activated fans), root zone temperature at 28°C (bed heaters) and 20 second misting at 10 minute intervals (automatic misters). The study was carried out in November 2005, April 2006 and June 2006. In the first experiment, both terminal and non-terminal cuttings were used; thereafter only non-terminal cuttings were used. The plantlet yield was very low, regardless of cutting type, Seradix 2 treatment and the time of year the cuttings were set. The highest plantlet production (12.5%) and rooting frequencies (13.8%) were achieved with non-terminal cuttings treated with Seradix 2. Although not statistically significant, Seradix 2 inhibited shoot production (31.4% for Seradix 2-untreated and 24.2% for treated cuttings). The position at which inserts were cut in relation to the node did not significantly affect the number of plantlets produced and non-terminal cuttings appeared hardier and performed better than terminal cuttings. The time of year of setting cuttings did not have any significant effect on plantlet yield, nonetheless, plantlet yield was highest in cuttings set in November (9.2%) and lowest in April (0.4%). In addition, cuttings set in November (spring), had superior shoot development in terms of the number of cuttings that produced shoots (regardless of root production), shoot length and the mass of shoots relative to root mass. The highest percentages of cuttings that produced roots (regardless of shoot growth) (10%) and the highest number of roots per cutting (2) were part of the June trial. Therefore, cuttings set in June (winter) had superior root development as compared with cuttings set in November (spring) or April (autumn). In all of the studies, three rooting patterns were observed in cuttings: roots produced only from the cut area only (type 1), only from the sides of the stem (type 2) and from both sites (type 3). Non-terminal cuttings treated with Seradix 2 showed a higher incidence of types 2 and 3 rooting patterns than the terminal cuttings. Seradix 2 application increased the prevalence of types 2 and 3 rooting patterns. Although not statistically different, cuttings dipped 2.5 cm into Seradix 2 produced more types 2 and 3 rooting patterns than cuttings dipped at the abaxial end only. Light microscopy of stem sections of cuttings indicated that roots appeared to originate from the xylem archs as well as from the cambium. The collected data indicate that it is necessary to continue research towards improving the efficiency of plantlet production of GN107 via cuttings. It appears that cuttings of this clone may be set throughout the year and that terminal cuttings should be avoided. In addition, the present practice at the Mondi Hilton nursery of treating cuttings with Seradix 2 needs to be reconsidered as although it increases rooting, it does not increase plantlet production due to its apparent inhibitory effect on shoot development. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2007.

Os agrupamentos de ferro-enxofre (Fe-S) são grupos prostéticos necessários para a manutenção da vida, pois estão envolvidos em diversos processos incluindo a transferência de elétrons, reações metabólicas, sinalização e regulação da expressão gênica. As plantas realizam fotossíntese e respiração, dois processos que requerem proteínas Fe-S, sendo os únicos organismos em que a síntese destas proteínas é compartimentalizada. Diversos fatores afetam o desenvolvimento das plantas, entre eles, a temperatura baixa, fator limitante à produtividade e à distribuição geográfica das plantas, incluindo Eucalyptus grandis, uma espécie com grande importância econômica. Neste trabalho foi realizada uma análise transcricional dos genes NFS1, ISA1 e ISU1 de E. grandis após diferentes estúmlos por meio de PCR quantitativa (qRT-PCR) e microarranjos. Após o tratamento de plântulas de E. grandis com frio, foram realizados experimentos de qRT-PCR. Os resultados foram normalizados com os genes constitutivos codificadores da histona H2B e da ribonucleoproteína L23A. Considerando tal normalização, ISU1 aumentou sua expressão em 0,6 e 1,7 vezes, NFS1 apresentou um aumento de 6 e 8 vezes, enquanto ISA1 apresentou um aumento de 69 a 114 vezes em relação à condição controle. Utilizando-se a técnica de microarranjos, foi analisada a diferença de expressão entre folhas e xilema de árvores maduras de E. grandis. O gene NFS1 apresentou maior expressão nas folhas do que em xilema, porém os genes ISA1 e ISU1 apresentaram um padrão de expressão equivalente entre os dois tipos de tecidos. Esses resultados sugerem que (i) os genes NFS1 e ISA1 podem estar relacionados à resposta celular ao estresse causado por frio; e que (ii) os aumentos na expressão devem-se, provavelmente, ao metabolismo de enxofre e à indução de enzimas antioxidantes. Foi também realizado um experimento de curva de tempo com a submissão de plântulas de E. grandis ao resfriamento, objetivando-se verificar em que momento esses genes começam a ter suas expressões aumentadas. O gene ISU1 apresentou maior expressão gênica nas primeiras duas horas de tratamento, caindo drasticamente logo após este período. O gene ISA1, que havia apresentado a maior expressão relativa no experimento anterior, não apresentou diferença significativa no padrão de expressão durante as 16 horas de resfriamento, assim como o gene NFS1. Esses resultados indicam que as proteínas Fe-S, frente ao resfriamento, estão possivelmente envolvidas na recuperação das plantas após tal estresse. / Iron-sulfur (Fe-S) clusters are prosthetic groups required for the maintenance of life because they are involved in various vital processes, including electron transfers, metabolic reactions, signaling and regulation of gene expression. Plants perform photosynthesis and respiration, two processes that require Fe-S proteins, and are the only organisms in which the synthesis of these proteins is compartmentalized. Several factors and stresses affect the development of plants including low temperature, which is a productivity-limiting factor and restricts plants to certain geographical distributions, including Eucalyptus grandis, a species with significant economic importance. The aim of this study is to perform an analysis of E. grandis NFS1 and ISA1 gene expressions after different stimuli through quantitative PCR (qRT-PCR) and microarrays. qRT-PCR experiment were conducted on plants submitted to a cold treatment. The results were normalized with the housekeeping genes encoding histone H2B and ribonucleoprotein L23A. Considering such normalizations, ISU1 increased the expression 0.6 and 1-fold, NFS1 showed a 6 and 8-fold increase in comparison with the control condition, while ISA1 gene increased 69 and 114-fold. Using microarrays, the difference in expression between leaves and xylem of E. grandis was analyzed. The NFS1 gene showed higher expression in leaves than in xylem, but the ISA1and ISU1 showed equivalent pattern of expression in both types of tissues. These results suggest that (i) NFS1 and ISA1 genes are related to the cellular response to the stress caused by chilling, and that (ii) the increased expression should be probably due to the metabolism of sulfur and to the induction of antioxidative enzymes. A time-course experiment was also conducted during the cold stress of E. grandis plants to look at which moment these genes begin to increase their expressions. The ISU1 gene showed higher expression in the first 2 hours of treatment, and than decreased severally after this period. The ISA1 gene, which had shown the highest expression in the previous experiment, did not show significant differences in the pattern of expression during the 16 hours of chilling treatment, as well as the NFS1 gene. These results indicate that Fe-S proteins, in response to low temperature, are possibly involved in the recovery of the plants after this stress.