It is believed that the process-based model 3-PG (Physiological Principles Predicting Growth; Landsberg and Waring, 1997) can potentially play a useful role within South African forestry, both as an operational and a strategic tool. Strategic applications may include the prediction of potential productivity on a site-by-site basis; broadscale productivity estimates based on remote sensing and the spatial application of 3-PG; identification of production constraints; and estimation of carbon fluxes to help address sustainability issues. Operationally, 3-PG could complement empiricallybased models or be used in conjunction with them as a hybridised product. The challenges of this study were therefore to see whether it is possible to adapt 3-PG to predict the growth and yield of E. grandis under South African conditions, test that model predictions are consistent with observed growth data and are biologically reasonable, and to assess the practicality of using 3-PG as either a strategic or operational tool. The main emphasis of this study was to understand the internal logic of 3-PG and how physiological processes are represented, and to develop methods to objectively parameterise and initialise the model. Thereafter a detailed model validation study was performed, ending off with selected potential applications of 3-PG within the South African context. The sensitivity of predicted stand volume (SV) and leaf area index (LAI) to the values of the species-specific parameters in 3-PG was examined. These analyses enabled the development of three distinct parameter sensitivity classes: insensitive parameters (i.e. those that can be varied widely without affecting the outputs studied), sensitive parameters (i.e. those whose value strongly affects the outputs, and non-linear parameters (i.e. those for which the outputs depend in a non-linear manner on the parameter value). Minimum data requirements for the parameterisation and initialisation of 3-PG are considered in detail. Conventional methods used for the parameterisation of models, specifically 3-PG, are reflected upon. An automated parameter estimation technique was examined and used for the estimation of selected parameters. Species-specific parameters were categorised according to data source estimation and sensitivity classes. Parameters describing allometric and age-dependent relationships were assigned values using observed data from biomass harvests. Critical parameters that could not be directly assigned using observed data were the ratio of foliage to stem allocation (i.e. P2 and p2o), allocation of net primary production (NPP) to roots (TJRX and T]Rn), optimum temperature for growth (7^,) and maximum canopy quantum efficiency (acx)- These were estimated using Parameter ESTimation, by fitting model output to corresponding observed growth data. As well as species-specific parameter values, mandatory inputs required by 3-PG include weather data, site-specific factors such as site fertility (FR) and physical properties of the soils, and stand initialisation data. Objective techniques to determine these site-specific factors and the initial values for the biomass pools were proposed. Most of these data are readily available for sites where experimental trials exist, or where monitoring networks are in place. However, this is the exception rather than the rule, so alternative data and information sources are required. These, together with the need for accurate weather inputs (especially monthly rainfall) and physical properties (especially soil texture, maximum available soil water and FR) of the sites being modelled were explored. 3-PG was validated using four simple tests by comparing predicted versus observed SV. Results showed that 3-PG predictions are relatively consistent with observed stand data. Analyses performed using time-series data showed model predictions accurately tracked observed growth in response to erratic and fluctuating weather conditions. Results from the initial model validation showed production on high and low productivity sites was under- and over-predicted, respectively. Further results presented here show a similar, but less marked trend (i.e. over- and under-predictions are not as extreme), and individual biases are less than those from predictions made using another locally developed parameter set. The application of 3-PG showed that the model is able to make estimates of tree growth that are consistent with those used within the forestry site classification. This showed the considerable potential 3-PG has for strategic planning by the forest industry (i.e. projected wood supplies etc) and in research planning (refining existing site classifications). The model could be useful in predicting growth in various areas where E. grandis is not grown, assisting in future decision making. 3-PG was able to identify growth constraints on a site-by-site basis and distinguish among them, and was able to identify environmental and site limitations to plantation growth, and how they vary in space and time. These results together with predictions of site productivity demonstrate the potential for 3-PG to be used to improve existing forest site classifications. The model comparison study between empirically-based models and 3-PG showed that although the empirical models made accurate predictions of volume under static climatic conditions, under fluctuating weather conditions empirical estimates of volume were less accurate than those made with 3-PG. 3-PG can therefore be used operationally with minimum input data to predict growth using enumeration data. This is useful in saving time and cutting costs. The use of process-based models (PBMs) in general, and 3-PG in particular, needs to be "championed'' to the South African forest industry. This is necessary for two reasons. Firstly, the model and the manner with which it describes physiological processes of growth need to be explained in layman's terms. This will demonstrate how and why a process-based model can work better in a fluctuating environment than empirically based models. Secondly the comparison between 3-PG and the local empirical models needs to be presented as an example of how 3-PG can be applied on an operational basis. It is accepted that much convincing is still required. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2005.

In South Africa, Eucalyptus breeding programmes are aimed at the selection of fastgrowing varieties, with appropriate wood characteristics and/or resistance to pests and diseases. However, the slow growth rate, long generation time and heterozygosity of trees make this a difficult task. Such problems may be overcome by the adoption of a biotechnological approach for plant propagation and modification. Towards this end, the aims of this investigation were to establish protocols for the micropropagation of Eucalyptus grandis and for the Agrobacterium-mediated transformation and subsequent plant regeneration of this important species. The usefulness of transformed cells and/or tissues is dependent upon the availability of methods for their regeneration into plants. Consequently, methods for plant regeneration via indirect organogenesis from leaf discs and cell suspension cultures were investigated. Organogenic calli were produced from leaf explants on MS medium with 16 mg.1-1 &l:em•calcltrate, 20 g.I-I sucrose, 1mg.I-I NAA and 0.05 mg.1--<1 BA. Shoots were induced on MS medium containing 1 mg.1-1 ZEA and 0.2 mg.1-1 IAA, and subsequently rooted on medium containing MS nutrients supplemented with 1 mg.1- 1 IAA. Cell suspension cultures were established but not regenerated via indirect organogenesis. Additionally, various media were investigated in order to obtain somatic embryos from cell suspension cultures. The MS media supplemented with 30 g.1-1 sucrose, 12 mg.1- 1 ABA and/or 40 g.1-1 PEG were found to be most suitable, resulting in the production of embryoids; germination results are not available at this stage. In order to establish methods for the transformation of both leaf discs and cell suspension cultures of Eucalyptus, a triparental mating was performed between Escherichia coli pnT119 (donor), A. tumefaciens LBA4404 (recipient), and E. coli HBI0l::pRK2013 (helper), resulting in the transconjugant LBA4404 (pnT119); insertion of the pJIT119 plasmid was demonstrated using agarose gel electrophoresis. The transconjugant CS8C1 (pMP90) (pJIT119) was also used. Protocols for the transformation of both leaf discs and cell suspension cultures were established, and resulted in the production of putatively transformed calli which were GUS positive and with stood selection on kanamycin (50 Ilg.mr1) and/or sulfadiazine (50 Ilg.mr1). Also, Southern blotting analysis indicated that the gene transfer process was successful. Due to difficulties in the regeneration of plants from transformed calli transgenic plants were not obtained. Future research strategies and applications of the developed protocols to Eucalyptus breeding programmes are discussed. / Thesis (M.Sc.)-University of Natal, 1994.

South Africa depends largely on afforestation programs for its timber supplies due to the great demands for fiber and wood products. This has brought discomfort to other water users who have advocated that the effects of afforestation on water resources are detrimental to the country as a whole since South Africa is known as a water scarce country. This study has undertaken to integrate a process-based model and remote sensing data to estimate water use and productivity of Eucalyptus grandis in the Zululand areas of South Africa. The remote sensing techniques and recently developed "process based model" that is 3PG-S were used to estimate water use and productivity of Eucalyptus grandis, an economically important plantation species grown in the summer rainfall areas of South Africa. The study utilized monthly Landsat Thematic Mapper datasets and climatic data as inputs into the 3PG-S model, determined the Leaf Area Index (LAI) and Specific Leaf Area (SLA) through direct (destructive sampling) and indirect measurements (LiCor- 2000) and assessed the relationships between various vegetation indices (VI's) using correlation and regression analyses. The results suggest that all the indices, except the ratio VI, correlated significantly with LiCor-determined and destructively measured LAI values with both normalized difference vegetation index (NDVI) and Ratio Vegetation Index (RVI) (r=0.86, p / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritburg, 2007.

Eucalypts are a major forest resource globally and the area of eucalypt plantations for pulp and paper production is expanding rapidly in Australia. Consequently, there is an increasing need to breed eucalypts with improved wood properties. Since many high value wood traits are under strong genetic control, identification of DNA markers linked to these traits will have application in breeding programs. In recent years there has been a shift in marker strategy away from QTL mapping in pedigrees to association studies in unrelated populations. In the latter approach, single nucleotide polymorphisms (SNPs) in candidate genes are screened to identify SNPs that significantly associate with wood traits. Significant SNPs could be used for marker-assisted selection (MAS) in breeding programs. The objectives of this study were to identify candidate genes that may influence pulp yield in eucalypts and to identify SNP variants in those genes that associate with superior wood and pulp traits. / Approximately 300 trees from a full-sib Eucalyptus nitens progeny derived from a wide intra specific cross were used for gene discovery. DNA microarrays containing ~5800 young xylem of cDNAs Eucalyptus grandis were screened with probes synthesised from RNA isolated from trees with either high or low pulp yield. Forty-six transcripts were differentially regulated, of which 27 were more abundant in high pulp trees and 19 were more abundant in low pulp trees. All differentially expressed cDNAs were partially sequenced and searched against existing gene databases. Six genes were selected as putative pulp yield candidate genes based on their significant similarity to genes with known function and were named EgrCesA3 (cellulose synthase), EgrNAM1 (NAM family protein), EgrXET (xyloglucan endotransglycosylase), EgrGalk (galactokinase), EgrHB1 (class III homeodomain leucine zipper protein) and EgrZnf1 (C3HC4 type zinc finger protein). / Real-Time PCR was carried out on selected genes to confirm the accuracy of the microarray results. Full length cDNAs were obtained for EgrCesA3, EgrHB1 and EgrZnf1 and the candidate genes were partially characterised. An additional candidate gene, the novel gene EgrPAAPA, was selected based on previous research due to its high expression in the cambium and its expression in eucalypt branches. EgrPAAPA was cloned by screening an E. grandis cDNA library and fully sequenced. The full length EgrPAAPA encodes a short 172 amino acid protein rich in alanine, glutamic acid and proline residues. The EgrPAAPA protein appears to be a hydroxyproline-rich glycoprotein (HRGP) and the repetitive ‘PAAPA’ motif suggests that it might play a structural role in cell wall development. Southern blot analysis revealed that E. grandis has a single copy of the EgrPAAPA gene and northern blot analysis revealed that EgrPAAPA is most strongly expressed in xylem tissues. / Allelic variation in EnCesA3, EnNAM1, EnPAAPA and EnHB1 was examined by sequencing each gene in 16 to 24 unrelated E. nitens individuals. SNPs were identified by sequence analysis and patterns of nucleotide diversity, linkage disequilibrium and the selection of suitable polymorphisms were estimated. A moderate level of nucleotide diversity (θw = 0.0056 and π = 0.0039) was observed and linkage disequilibrium was generally low, extending only a few hundred base pairs in each gene. Negative selection has been operating in EnHB1. Selected TagSNPs from EnNAM1, EnHB1 and EnPAAPA were genotyped across 300 unrelated E. nitens trees which had been phenotyped for six wood quality traits including pulp yield, cellulose, lignin, Klason lignin, microfibril angle (MFA) and density. Five highly significant genetic associations (p<0.01) were detected between several SNPs in EnHB1 and all wood quality traits except density. A significant association was also found between EnPAAPA and MFA (p<0.05). No significant associations were found with any of the EnNAM1 SNPs. The strong genetic associations between SNPs in EnHB1 and a range of wood traits is consistent with this gene’s known role as a transcription factor controlling vascular development. Validation of these associations in different populations will be necessary in order to confirm these results. Alternatively, QTL mapping can be performed in order to confirm whether QTL for wood property traits can be detected at the EnHB1 and EnPAAPA loci.

Thesis (M.S.)--Oregon State University, 1995. / Typescript (photocopy). Includes bibliographical references (leaves 79-82). Also available on the World Wide Web.

Orientador: Claudio Angeli Sansígolo / Banca: Elias Taylor Durgante Severo / Banca: Ezer Dias de Oliveira Junior / Banca: Tatiana Paula Marques de Arruda / Banca: Edjair Augusto Dal Bem / Resumo: A indústria papeleira vem focando em estudos atuais, alternativas para separar a madeira em seus componentes, celulose, hemiceluloses, lignina e extrativos, pois eles que restringem as diversas oportunidades da biorrefinaria integrando processos produtivos de combustíveis e produtos químicos a partir da biomassa. A solubilização das hemiceluloses por auto hidrólise tem sido proposta como o primeiro passo da biorrefinaria, e os materiais extraídos com a auto hidrólise podem ser usados na produção de produtos químicos e biocombustíveis. Assim, este trabalho avaliou o efeito da temperatura (120°C,140°C,160°C) na auto hidrólise, e posteriormente no rendimento da polpação Kraft em diferentes cargas de álcali ativo (12%,13%,14%) em cavacos de Eucalyptus grandis x Eucalyptus urophylla e Eucalyptus grandis. Os resultados da pesquisa mostraram que com o incremento da temperatura no processo de auto hidrólise o rendimento apresenta alteração para ambas espécies; reações com maiores valores de temperaturas afetam negativamente o rendimento do processo de auto hidrolise; a utilização da Eucalyptus grandis mostrou mais eficaz no processo de auto hidrolise quando comparado ao híbrido E. grandis x E. urophylla. Conclui-se que, a auto hidrólise apresentou eficácia removendo a maior parte das hemiceluloses e apenas atacando ligeiramente a lignina e a celulose. / Abstract: The paper industry has been focusing on current studies alternatives for separating wood into its components, cellulose, hemicellulose, lignin and extractives, as they that hide the various biorefinery opportunities integrating production processes of fuels and chemicals from biomass. The solubilization of hemicelluloses by auto-hydrolysis has been proposed as the first step in biorefinery. And the materials extracted with the Auto-hydrolysis can be used in the production of chemicals and biofuels. Thus, this study evaluated the effect of temperature (120 ° C, 140 ° C, 160 ° C) in the auto-hydrolysis and Kraft pulping yield in different loads of active alkali (12%, 13%, 14%) in Eucalyptus grandis x Eucalyptus urophylla e Eucalyptus grandis chips. The survey results showed that with the increase of temperature in the process of hydrolysis yield self presents to both species; reactions with highest rates of temperatures affect negatively the performance of the hydrolysis process; the use of Eucalyptus grandis showed more effective in hydrolysis process when compared the hibrid E. grandis x E. urophylla and concluded that, the hydrolysis showed effectiveness by removing most of the hemicelluloses and attacking only slightly the lignin and cellulose ... / Doutor

Orientador: Luiz Gonzaga de Souza / Resumo: O presente trabalho de pesquisa tem por objetivo estimar a composição dos resíduos e o volume de biomassa disponível para fins energéticos da exploração de Eucalyptus grandis em uma indústria de painéis de fibra de madeira (hardboard) localizada no Município de Botucatu, Estado de São Paulo. Para isso foram coletadas amostras no campo, referentes ao resíduo deixado por quinze árvores de Eucalyptus na área correspondente a um hectare, sendo estimados nas amostras o poder calorífico superior (PCS), teor de umidade, teor de cinzas, densidade básica e volume total médio da biomassa residual. Foi estimado, ainda, o vapor obtenível por quilograma de madeira queimada para avaliação preliminar de energia elétrica a ser produzida por tonelada de biomassa (kWh/tb), fornecendo subsídios para estudos futuros de viabilidade econômica de um projeto de cogeração ou geração própria de energia elétrica. Os resultados obtidos mostram os valores médios de poder calorífico superior da biomassa coletado no campo: 4001, 3907, 3845, e 3704, kcal/kg para amostras (ramos) de até 1 cm de diâmetro, de 1 a 3 cm de diâmetro, acima de 3 cm de diâmetro e casca, respectivamente. O volume total médio de biomassa residual produzido foi de 16,167 m3/ha com um peso estimado de 8375 kg/ha. Os resultados obtidos para a quantidade de vapor obtida por quilograma de madeira queimada (fator f) e o peso total de vapor fornecido pela biomassa residual foi da ordem de 36.599 kg vapor/ ha. / Abstract: The present research aimed to determine the waste composition and the biomass volume available to energetic exploitation of Eucalyptus grandis in a hardboard industry located in Botucatu, São Paulo State. Field samples were collected in the waste of fifteen Eucalyptus grandis trees, in a area equivalent of one hectare. In each sample were evaluated the heat combustion , the humidity content, ash content, basic density and the total mean volume of waste biomass. It was also analyzed achieved steam per kilo of burned wood to a initial evaluation of the electrical energy to be produced by each ton of biomass (kW/tb), offering subsidies to future economic availability studies of an own electrical energy generation project. The obtained data showed that the heat combustion of the wood varied from 4021, 3907, 3845 and 3704 kcal/kg to samples (branchs) of one centimeter, from one to three centimeters and to more than three centimeters of diameter and bark, respectively. The total mean volume of the waste biomass produced was 16.167 m3/ha with an estimated weight of 8375 kg/ha. The results obtained to the steam quantity obtained per each kilo of burned wood (f) and the total steam weight given by the waste biomass were 36.599,49 kg steam/ha. / Mestre

Uma cuidadosa revisão bibliográfica, seguida por uma pesquisa in silico no banco de dados de expressed sequence tags (ESTs) do projeto GENOLYPTUS – Rede Brasileira de Pesquisa do Genoma de Eucalyptus, foram realizadas visando identificar classes de genes potencialmente relacionadas com a resposta vegetal à deficiência hídrica em Eucalyptus grandis. Os principais clones de cDNA selecionados potencialmente codificam proteínas relacionadas com a síntese de osmólitos como prolina, sacarose, osmotina, poliaminas, desidrinas, fatores de transcrição, e genes de uma importante cascata de sinalização vegetal à deficiência hídrica. Duas classes de seqüências foram estudadas mais detalhadamente. A Sadenosil- metionina-descarboxilase (SAMDC) é uma enzima-chave na rota de síntese das poliaminas, aminas catiônicas envolvidas em vários processos celulares, inclusive na proteção das estruturas da célula em respostas a diferentes tipos de estresses. Dois grupos de ESTs foram inicialmente identificados por homologia a seqüências conhecidas de SAMDC e duas seqüências potencialmente completas de cada grupo foram selecionadas e denominadas EgrSAMDC1 e EgrSAMDC2. As seqüências deduzidas de aminoácidos de ambos os cDNAs apresentaram os dois principais domínios da enzima e uma estrutura conservada em relação a outras SAMDCs de plantas. Além da região de leitura aberta (ORF, do inglês, open reading frame) principal, os cDNAs apresentaram duas pequenas regiões codificadoras conservadas, denominadas tiny e small upstream ORFs na longa região 5’ não traduzida, relacionadas à regulação pós-transcricional dos genes. Em análises de Southern blot, foram detectados pelo menos dois fortes sinais de hibridização para cada amostra de DNA genômico de E. grandis digerido por endonucleases distintas, sugerindo a existência de (pelo menos) duas cópias genômicas de SAMDC. Por Northern blots, a presença de mRNAs para SAMDC foi detectada em folhas e caules jovens e, em menor quantidade, em tecidos vasculares de árvores adultas. Entretanto e nitidamente, a maior expressão de SAMDC em E. grandis ocorre em flores abertas. Análises quantitativas por transcrição reversa seguida de reação em cadeia da DNApolimerase (qRT-PCR) com plântulas cultivadas in vitro por 4 meses, submetidas a diferentes tratamentos como ácido abscísico (ABA), ácido naftalenoacético (ANA), quinetina (KIN), cloreto de sódio (NaCl), seca, frio, luz ultravioleta (UV), ferimentos e extrato de leveduras, 9 demonstraram maior indução da expressão de EgrSAMDC2 na presença de ABA e NaCl. Também foi observada uma drástica redução na expressão deste gene, baseado nos níveis de mRNA-molde para as RT-PCRs, quando as plântulas foram expostas a UV e seca. Estes resultados indicaram que o gene EgrSAMDC2 pode estar mais relacionado à resposta vegetal à elevada salinidade, por intermédio da sinalização por ABA, do que à resposta a outros estresses ambientais ou bióticos. A segunda classe de genes identificados e analisados junto ao GENOLYPTUS como potencialmente relacionados à deficiência hídrica potencialmente codificam proteína-cinases ativadas por mitógenos (MAPKs, do inglês, mitogen-activated protein kinases). MAPKs são proteínas de uma importante cascata sinalizadora, constituída por três componentes denominados MAPKKK, MAPKK e MAPK. Elas estão envolvidas na sinalização do desenvolvimento, estímulos hormonais e de estresses bióticos e abióticos. O cDNA selecionado, de aproximadamente 5 kb, representa uma seqüência imatura de mRNA (transcrito primário), no qual estão presentes dois íntrons. Nas extremidades 5’ e 3’ dos íntrons existem sítios canônicos de processamento (splicing) que indicam que o transcrito deve ser corretamente processado em um mRNA maduro. A proteína deduzida a partir da união dos éxons indica que o cDNA isolado de E. grandis potencialmente codifica uma MAPK2, com alta homologia a outras MAPKs vegetais. Além disto, a seqüência de aminoácidos representa uma proteína completa, na qual os 11 domínios conservados em MAPKs, fundamentais para o correto funcionamento da enzima, estão presentes. Análise de Southern blot indicaram que este gene está presente em cópia única no genoma de E. grandis. Em análises da expressão por qRT-PCR em plântulas submetidas aos diferentes tratamentos descritos acima, foi observado uma significativa indução da transcrição com KIN e NaCl, e uma leve indução por ABA. O tratamento com UV provocou uma drástica redução na expressão de EgrMAPK2. Este resultado fornece indícios de que este gene possivelmente esteja em envolvido em repostas a estresses abióticos. / A careful bibliographical review, followed by an in silico screening of the expressed sequence tag (EST) databank of the GENOLYPTUS Project – The Brazilian Research Network on the Eucalyptus Genome, were conducted to identify genes potentially related to drought responses in Eucalyptus grandis. The main selected clones potentially encode proteins related to the biosynthesis of different osmolites such as proline, sucrose, osmotin and polyamines, dehydrines, transcription factors and genes from an important plant signaling cascade. Two gene classes were studied in more detail. S-adenosylmethionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines, cationic amines involved in various processes of the cell, including the protection of cellular structures in response to different stresses. Two clusters of ESTs were initially identified by homology to known SAMDC sequences and, from each of them, two full-length cDNAs were identified and named EgrSAMDC1 and EgrSAMDC2. The deduced amino acid sequences from both cDNAs showed the two main enzyme domains and a conserved protein structure in comparison to other plant SAMDCs. Besides the main open reading frame (ORF), two other tiny and small upstream ORFs were found in the long 5’ non-translated region of the cDNAs. These sequences are known to be associated with post-transcriptional regulation. Southern blot analysis resulted in two strong hybridization signals for each genomic DNA digested with different restriction enzymes, indicating (at least) two SAMDC genomic copies. Northern blots detected SAMDC mRNAs in young leaves and stems and a lower transcript level in vascular tissues from mature trees. Nevertheless, the highest levels of E. grandis SAMDC steady-state mRNA was clearly detected in opened flowers. Quantitative reverse transcription PCR (qRT-PCR) with total RNA from 4-months old in vitro seedlings subjected to different treatments, such as abscisic acid (ABA), naphthaleneacetic acid (NAA), kinetin (KIN), sodium chloride (NaCl), drought, cold, ultraviolet light (UV), wounding and yeast extract, demonstrated higher expression induction of EgrSAMDC2 gene with ABA and NaCl. A drastic reduction in gene expression was observed after UV and drought treatments of the seedlings, based on RNA-template levels for RT-PCRs. These results suggested that 11 EgrSAMDC2 gene might be more related to plant response against high salinity, possibly via ABA signaling, than to other environmental or biotic-like stress responses. The second gene class identified and analyzed in the GENOLYPTUS, as potentially involved in drought responses, encodes a mitogen-activated protein kinase (MAPK). MAPKs proteins are part of an important signaling casacade, which consists of three components, MAPKKK, MAPKK and MAPK. They are associated to developmental signaling, hormonal and biotic and abiotic stimuli. The selected cDNA, of about 5 kb in length, represented an immature mRNA (primary transcript) with two non-spliced introns. The canonical 5’ and 3’ splicing sites suggested that the transcript might probably be correctly translated to a mature mRNA. The deduced protein from the union of all three exons indicated that the isolated E. grandis cDNA potentially encodes a MAPK2, with high homology to other plant MAPKs. Moreover, the amino acid sequence represented a complete protein, with all 11 domains conserved in MAPKs and known to be fundamental for the enzyme functionality. Southern blot analysis indicated that EgrMAPK2 is encoded by a single genomic copy in E. grandis. From qRT-PCR expression analysis, conducted with the same plant material described above, we observed a significant transcription induction with KIN and NaCl, and a weaker induction by ABA. The UV treatment resulted in a drastic reduction of EgrMAPK2 expression. This result strongly indicated that this gene is most probably involved in abiotic stress responses.

Uma cuidadosa revisão bibliográfica, seguida por uma pesquisa in silico no banco de dados de expressed sequence tags (ESTs) do projeto GENOLYPTUS – Rede Brasileira de Pesquisa do Genoma de Eucalyptus, foram realizadas visando identificar classes de genes potencialmente relacionadas com a resposta vegetal à deficiência hídrica em Eucalyptus grandis. Os principais clones de cDNA selecionados potencialmente codificam proteínas relacionadas com a síntese de osmólitos como prolina, sacarose, osmotina, poliaminas, desidrinas, fatores de transcrição, e genes de uma importante cascata de sinalização vegetal à deficiência hídrica. Duas classes de seqüências foram estudadas mais detalhadamente. A Sadenosil- metionina-descarboxilase (SAMDC) é uma enzima-chave na rota de síntese das poliaminas, aminas catiônicas envolvidas em vários processos celulares, inclusive na proteção das estruturas da célula em respostas a diferentes tipos de estresses. Dois grupos de ESTs foram inicialmente identificados por homologia a seqüências conhecidas de SAMDC e duas seqüências potencialmente completas de cada grupo foram selecionadas e denominadas EgrSAMDC1 e EgrSAMDC2. As seqüências deduzidas de aminoácidos de ambos os cDNAs apresentaram os dois principais domínios da enzima e uma estrutura conservada em relação a outras SAMDCs de plantas. Além da região de leitura aberta (ORF, do inglês, open reading frame) principal, os cDNAs apresentaram duas pequenas regiões codificadoras conservadas, denominadas tiny e small upstream ORFs na longa região 5’ não traduzida, relacionadas à regulação pós-transcricional dos genes. Em análises de Southern blot, foram detectados pelo menos dois fortes sinais de hibridização para cada amostra de DNA genômico de E. grandis digerido por endonucleases distintas, sugerindo a existência de (pelo menos) duas cópias genômicas de SAMDC. Por Northern blots, a presença de mRNAs para SAMDC foi detectada em folhas e caules jovens e, em menor quantidade, em tecidos vasculares de árvores adultas. Entretanto e nitidamente, a maior expressão de SAMDC em E. grandis ocorre em flores abertas. Análises quantitativas por transcrição reversa seguida de reação em cadeia da DNApolimerase (qRT-PCR) com plântulas cultivadas in vitro por 4 meses, submetidas a diferentes tratamentos como ácido abscísico (ABA), ácido naftalenoacético (ANA), quinetina (KIN), cloreto de sódio (NaCl), seca, frio, luz ultravioleta (UV), ferimentos e extrato de leveduras, 9 demonstraram maior indução da expressão de EgrSAMDC2 na presença de ABA e NaCl. Também foi observada uma drástica redução na expressão deste gene, baseado nos níveis de mRNA-molde para as RT-PCRs, quando as plântulas foram expostas a UV e seca. Estes resultados indicaram que o gene EgrSAMDC2 pode estar mais relacionado à resposta vegetal à elevada salinidade, por intermédio da sinalização por ABA, do que à resposta a outros estresses ambientais ou bióticos. A segunda classe de genes identificados e analisados junto ao GENOLYPTUS como potencialmente relacionados à deficiência hídrica potencialmente codificam proteína-cinases ativadas por mitógenos (MAPKs, do inglês, mitogen-activated protein kinases). MAPKs são proteínas de uma importante cascata sinalizadora, constituída por três componentes denominados MAPKKK, MAPKK e MAPK. Elas estão envolvidas na sinalização do desenvolvimento, estímulos hormonais e de estresses bióticos e abióticos. O cDNA selecionado, de aproximadamente 5 kb, representa uma seqüência imatura de mRNA (transcrito primário), no qual estão presentes dois íntrons. Nas extremidades 5’ e 3’ dos íntrons existem sítios canônicos de processamento (splicing) que indicam que o transcrito deve ser corretamente processado em um mRNA maduro. A proteína deduzida a partir da união dos éxons indica que o cDNA isolado de E. grandis potencialmente codifica uma MAPK2, com alta homologia a outras MAPKs vegetais. Além disto, a seqüência de aminoácidos representa uma proteína completa, na qual os 11 domínios conservados em MAPKs, fundamentais para o correto funcionamento da enzima, estão presentes. Análise de Southern blot indicaram que este gene está presente em cópia única no genoma de E. grandis. Em análises da expressão por qRT-PCR em plântulas submetidas aos diferentes tratamentos descritos acima, foi observado uma significativa indução da transcrição com KIN e NaCl, e uma leve indução por ABA. O tratamento com UV provocou uma drástica redução na expressão de EgrMAPK2. Este resultado fornece indícios de que este gene possivelmente esteja em envolvido em repostas a estresses abióticos. / A careful bibliographical review, followed by an in silico screening of the expressed sequence tag (EST) databank of the GENOLYPTUS Project – The Brazilian Research Network on the Eucalyptus Genome, were conducted to identify genes potentially related to drought responses in Eucalyptus grandis. The main selected clones potentially encode proteins related to the biosynthesis of different osmolites such as proline, sucrose, osmotin and polyamines, dehydrines, transcription factors and genes from an important plant signaling cascade. Two gene classes were studied in more detail. S-adenosylmethionine decarboxylase (SAMDC) is one of the key regulatory enzymes in the biosynthesis of polyamines, cationic amines involved in various processes of the cell, including the protection of cellular structures in response to different stresses. Two clusters of ESTs were initially identified by homology to known SAMDC sequences and, from each of them, two full-length cDNAs were identified and named EgrSAMDC1 and EgrSAMDC2. The deduced amino acid sequences from both cDNAs showed the two main enzyme domains and a conserved protein structure in comparison to other plant SAMDCs. Besides the main open reading frame (ORF), two other tiny and small upstream ORFs were found in the long 5’ non-translated region of the cDNAs. These sequences are known to be associated with post-transcriptional regulation. Southern blot analysis resulted in two strong hybridization signals for each genomic DNA digested with different restriction enzymes, indicating (at least) two SAMDC genomic copies. Northern blots detected SAMDC mRNAs in young leaves and stems and a lower transcript level in vascular tissues from mature trees. Nevertheless, the highest levels of E. grandis SAMDC steady-state mRNA was clearly detected in opened flowers. Quantitative reverse transcription PCR (qRT-PCR) with total RNA from 4-months old in vitro seedlings subjected to different treatments, such as abscisic acid (ABA), naphthaleneacetic acid (NAA), kinetin (KIN), sodium chloride (NaCl), drought, cold, ultraviolet light (UV), wounding and yeast extract, demonstrated higher expression induction of EgrSAMDC2 gene with ABA and NaCl. A drastic reduction in gene expression was observed after UV and drought treatments of the seedlings, based on RNA-template levels for RT-PCRs. These results suggested that 11 EgrSAMDC2 gene might be more related to plant response against high salinity, possibly via ABA signaling, than to other environmental or biotic-like stress responses. The second gene class identified and analyzed in the GENOLYPTUS, as potentially involved in drought responses, encodes a mitogen-activated protein kinase (MAPK). MAPKs proteins are part of an important signaling casacade, which consists of three components, MAPKKK, MAPKK and MAPK. They are associated to developmental signaling, hormonal and biotic and abiotic stimuli. The selected cDNA, of about 5 kb in length, represented an immature mRNA (primary transcript) with two non-spliced introns. The canonical 5’ and 3’ splicing sites suggested that the transcript might probably be correctly translated to a mature mRNA. The deduced protein from the union of all three exons indicated that the isolated E. grandis cDNA potentially encodes a MAPK2, with high homology to other plant MAPKs. Moreover, the amino acid sequence represented a complete protein, with all 11 domains conserved in MAPKs and known to be fundamental for the enzyme functionality. Southern blot analysis indicated that EgrMAPK2 is encoded by a single genomic copy in E. grandis. From qRT-PCR expression analysis, conducted with the same plant material described above, we observed a significant transcription induction with KIN and NaCl, and a weaker induction by ABA. The UV treatment resulted in a drastic reduction of EgrMAPK2 expression. This result strongly indicated that this gene is most probably involved in abiotic stress responses.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-04T18:25:44Z No. of bitstreams: 1 texto completo.pdf: 360867 bytes, checksum: e3ee01960616eb812f4e14a90f89fff5 (MD5) / Made available in DSpace on 2017-04-04T18:25:44Z (GMT). No. of bitstreams: 1 texto completo.pdf: 360867 bytes, checksum: e3ee01960616eb812f4e14a90f89fff5 (MD5) Previous issue date: 1998-11-27 / Plantas de Eucalyptus grandis foram aclimatadas a 25 o C e 400 μmol fótons m -2 s -1 , em câmara de crescimento, e posteriormente submetidas a uma combinação de temperatura e irradiância supra-ótimas, 40 o C e 1.200 μmol fótons m -2 s -1 , a fim de se avaliarem as respostas fisiológicas dessa espécie a uma condição potencialmente fotoinibitória. Não se observaram efeitos isolados da temperatura ou do excesso de irradiância sobre a eficiência fotoquímica do Fotossistema II (FS II), avaliada como a razão entre a fluorescência variável e a fluorescência máxima (F v /F m ), porém observou-se um efeito sinérgico desses dois fatores. A redução de F v /F m foi atribuída, primariamente, à redução da fluorescência máxima (F m ). A pré-aclimatação dos discos foliares à temperatura de 35 o C e baixa irradiância, 5 μmol fótons m -2 s -1 , por duas horas, resultou em aumento da tolerância à condição fotoinibitória. Pode-se inferir que a aquisição de tolerância à fotoinibição do PS II de E. grandis envolveu, primariamente, a alteração conformacional do centro de reação do PS II. Não se pode descartar o envolvimento da síntese de novo de proteínas neste processo, em razão do tempo requerido para se observar a alteração. O funcionamento de alguns componentes do sistema antioxidativo foi avaliado no sistema Eucalyptus-Pisolithus, em resposta à elevação da temperatura de 25 o C-28 o C para 40 o C. As alterações nas atividades de catalase (Cat), peroxidases não-específicas (Pod) e superóxido dismutase (Sod), em dois isolados fúngicos, traduziram estratégias diferentes de regulação do sistema antioxidativo. Houve reduções significativas nas atividades específicas da Cat, das Pod e da ascorbato peroxidase (AscPod), da atividade total da Sod e do teor de proteínas solúveis em folhas de E. grandis, após incubação a 40 o C. Essas alterações refletiram no menor teor de clorofila a e no aumento do teor de malondialdeído, uma indicação de que houve peroxidação de lipídios. Em raízes, não se observou efeito significativo da temperatura sobre as atividades de Cat, Pod e Sod. Não se detectou atividade da AscPod nesse órgão. Houve redução significativa do teor de proteínas solúveis e aumento do teor de malondialdeído após incubação a 40 o C, por três horas. As relações Sod:Cat e Sod:Pod nas micorrizas seguiram o padrão de aumento ou de redução observado no micélio dos dois isolados fúngicos. A atividade total da Sod foi maior do que nas raízes, a despeito da sua menor atividade nos micélios fúngicos. Este aumento resultou da indução de 4 a 5 novas isoenzimas de Sod do tipo Mn-Sod, todas de baixo peso molecular e com possível origem fúngica. O conjunto dos resultados obtidos permite inferir que o controle do sistema antioxidativo é exercido de forma mais eficiente nas micorrizas do que nos componentes individuais da associação mutualista. Este controle pode ser um dos responsáveis pelo benefício mútuo decorrente da associação simbiótica, especialmente observado em condições ambientais pouco favoráveis à sobrevivência dos organismos que dela participam. / Plants of Eucalyptus grandis were acclimated at 25 o C and 400 μmol photons m -2 s -1 , in growth chamber, and thereafter submitted to a high temperature/irradiance of 40 o C and 1,200 μmol photons m -2 s -1 , in order to investigate the physiological response of this species to a potential photoinhibitory condition. Based on variable to maximum fluorescence ratio (Fv/Fm), isolated effects of both high temperature and irradiance upon the photochemical efficiency of photosystem II (PSII) were not observed, but a synergic effect of these parameters was found. The decline in Fv/Fm ratio resulted mainly from a quenched maximal fluorescence (Fm). By incubating the leaves at 35 o C and at a low irradiance of 5 μmol photons m -2 s -1 for two hours, a higher tolerance to the photoinhibitory treatment could be observed. Given the time required in the process of thermotolerance induction, the involvement of “de novo” protein synthesis can not be dismissed. Some components of the antioxidant system were evaluated in the Eucalyptus-Pisolithus association, in response to an increase in temperature from 25 o C-28 o C to 40 o C. The changes in the activity of catalase (Cat), non-specific peroxidases (Pod), and that of superoxide dismutase (Sod), in two fungal isolates, are evidences for different strategies of the antioxidant system regulation in these organisms. Significant decreases in the specific activity of Cat, Pod, and ascorbate peroxidase (AscPod), total activity of Sod and of soluble proteins were observed in leaves of E. grandis incubated at 40 o C. These changes were accompanied by a lower chlorophyll a and a higher malondialdehyde content. In the root system, significant effects of the temperature upon the activities of Cat, Pod, and Sod were not observed, and AscPod activity was not detected in that organ. A significant decrease in protein and a rise in malondialdehyde content were observed after incubation of the roots at 40 o C for three hours. The Sod:Cat and Sod:Pod ratios in mycorrhizas followed the pattern of increase or decrease observed in the mycelium of the two fungal isolates. The total activity of Sod in the mycorrhizas was higher than that of the roots, despite its lower activity in the mycelium. This increase was attributed to the induction of 4 to 5 new Sod isozymes of the Mn-Sod class, all of low molecular weight and possibly coded by the fungal genome. The results obtained in this work indicate that the control of the antioxidant system is exerted more efficiently in the mycorrhizas than in the individual components of the association. This control may be partly responsible for the mutual benefits resulting from the symbiotic association, especially under environmental conditions less favorable to the survival of the symbionts. / Tese importada do Alexandria