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The focus of the work has been on computational approaches to solving biological problems. First, microarray analysis was used to study the role of PLDα1 in drought stress in Arabidopsis. Second, a tool for designing and in-silico testing of primers for PCR-based pathogen detection will be discussed. Phospholipase D (PLD) has been implicated in a variety of stresses including osmotic stress and wounding. PLDα 1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and promotes abscisic acid signaling. Plants with abrogated PLDα 1 show insensitivity to ABA and impaired stomatal conductance. My goal is to identify PLDα-mediated downstream events in response to progressive drought stress in Arabidopsis. <i>Arabidopsis thaliana</i> (Col-0) and antisense-PLDα 1 (Anti-PLDα) were drought stressed by withholding water. Anti-PLDα experienced severe water stress at the same time period that Col-0 experienced less water stress. Diurnal leaf water potential (LWP) measurements showed that Anti-PLDα had lower LWP than Col-0 under drought stress conditions. qRT-PCR revealed up to 18-fold lower values for PLDα transcripts in stressed Anti-PLDα plants when compared to stressed Col-0. Microarray expression profiles revealed distinct gene expression patterns in Col-0 and Anti-PLDα. ROP8, PLDδ and lipid transfer proteins were among the differentially expressed genes between the two genotypes. Different microarray analyses methods (TM4 and Expresso) were also compared on two different data sets. The results obtained from Expresso analysis were more accurate when compared with quantitative RT-PCR data. Rapid diagnosis of disease-causing agents is extremely important since delayed diagnosis can result in disease spread and delayed prophylaxis. It is even more important in an era where disease-causing agents are used as bioterrorism agents. Rapid advances in sequencing technology have resulted in the sequencing of thousands of microorganisms in recent years. Availability of genomic sequences has made it possible to identify and characterize microorganisms at the molecular level. PCR-based detection is powerful for pathogen diagnostics since it is rapid and sensitive. We have developed a tool, PathPrime, that can design primers, computationally test them against target genes, and potential contaminant sequences, and identify a minimum set of primers that can unambiguously detect a given list of sequences. / Ph. D.

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Doctor of Philosophy / Department of Agronomy / P.V. Vara Prasad / Sorghum (Sorghum bicolor L. Moench) is the fifth most economically important cereal crop grown worldwide and adapted to a wide range of climatic conditions. Drought stress has been ranked as one of the most significant causes of crop yield loss with its effects on yield and yield components. Conservative water use by plants is one of the strategies that can be used as a drought coping mechanism. The slow wilting trait has been associated with conservative water use and has been found in some sorghum genotypes. The purpose of this study was to use canopy temperature to screen for drought tolerance in sorghum, evaluate water use efficiency for slow wilting sorghum genotypes and determine variability in root morphology and response to drought among sorghum genotypes. Canopy temperature studies were conducted under field conditions using infrared (IR) sensors while water use efficiency and root studies were conducted under greenhouse conditions. Our results showed a distinct separation in canopy temperature among genotypes under field conditions at 2:00 pm to 6:00 pm. Midday canopy temperature depression (CTD) was positively correlated to yield (R2 = 0.19) and harvest index (R2 = 0.11). CTD was also stable for all the genotypes during the period from 1:00 pm to 7:00 pm. There was a negative correlation between CTD and crop water stress index (CWSI) (R2 = 0.34) and a positive one between canopy temperature and CWSI (R2 = 0.50). Evaluation of genotypes for water use efficiency revealed significant variability among sorghum genotypes in the amount of water used (10.48 – 13.52 kg) and transpiration efficiency (TE) (2.64 – 7.11 g kg-1) among genotypes. Slow wilting genotypes were high in TE. Rooting depth increased for some genotypes under drought stress with genotype SC1124 recording the largest increase (180%). Total root length for some genotypes increased by 11 – 113% with genotypes SC224 and SC1019 recording the greatest increase. There was a positive correlation between water used and root length (R2 = 0.21). These results show that there is potential for selection of drought tolerance in sorghum and that genotypes with the slow wilting traits are efficient in water use.

Doctor of Philosophy / Department of Agronomy / P.V. Vara Prasad / High temperature (HT) and drought are detrimental to crop productivity, but there is limited variability for these traits among wheat ([italics]Triticum aestivum[end italics] L.) cultivars. Five [italics]Aegilops[end italics] species were screened to identify HT (52 accessions) and drought (31 accessions) tolerant species/accessions and ascertaining traits associated with tolerance. Four synthetic wheats were studied to quantify independent and combined effects of HT and drought. [italics]Aegilops[end italics] species were grown at 25/19°C day/night and 18 h photoperiod. At anthesis, HT was imposed by transferring plants to growth chambers set at 36/30°C, whereas in another experiment, drought was imposed by withholding irrigation. Synthetic wheats were grown at 21/15°C day/night and 18 h photoperiod. At anthesis or 21 d after anthesis, plants were exposed to optimum condition (irrigation + 21/15°C), HT (irrigation + 36/30°C), drought (withhold irrigation + 21/15°C), and combined stress (withhold irrigation + 36/30°C). Stresses were imposed for 16 d. High temperature and drought stress significantly decreased chlorophyll, grain number, individual grain weight, and grain yield of [italics]Aegilops[end italics] species (≥ 25%). Based on a decrease in grain yield, [italics]A. speltoides[end italics] and [italics]A. geniculata[end italics] were most tolerant (~ 61% decline), and [italics]A. longissima[end italics] was highly susceptible to HT stress (84% decline). Similarly, [italics]A. geniculata[end italics] had greater tolerance to drought (48% decline) as compared to other species (≥ 73% decline). Tolerance was associated with higher grains spike [superscript]-1 and/or heavier grains. Within [italics]A. speltoides[end italics], accession TA 2348 was most tolerant to HT with 13.5% yield decline and a heat susceptibility index (HSI) 0.23. Among [italics]A. geniculata[end italics], TA 2899 and TA 1819 were moderately tolerant to HT with an HSI 0.80. TA 10437 of [italics]A. geniculata[end italics] was the most drought tolerant accession with 7% yield decline and drought susceptibility index 0.14. Irrespective of the time of stress, HT, drought, and combined stress decreased both individual grain weight and grain yield of synthetic wheats by ≥ 37%, 26%, and 50%, respectively. These studies suggest a presence of genetic variability among [italics]Aegilops[end italics] species that can be utilized in breeding wheat for HT and drought tolerance at anthesis; and combined stress of drought and high temperature on synthetic wheats are hypo-additive in nature.

Plantas com o tipo fotossintético CAM podem suportar condições de baixa disponibilidade de água através de um sistema de concentração de gás carbônico que aumenta a eficiência de uso da água através de uma fixação inicial do CO2 durante a noite, em condições de baixo déficit de pressão de vapor entre a folha e a atmosfera. Esse processo favorece a ocupação de regiões áridas e de sítios onde a água pode se tornar rapidamente indisponível. A ocorrência desse tipo fotossintético abrange uma grande amplitude de táxons, incluindo a família Bromeliaceae. Entre as espécies de Bromeliaceae que apresentam CAM encontra-se Dickya tuberosa, uma espécie que apresenta uma grande abundancia nos afloramentos rochosos associados ao monumento natural da Pedra Grande - Atibaia - SP. Nesse local, D. tuberose apresenta um papel relevante na vegetação associada à superfície de rocha exposta como componente essencial das comunidades denominadas \"ilhas de vegetação\". As condições predominantes no afloramento rochoso da Pedra Grande são de grande escassez de solo e água, além de extrema exposição. As variações na disponibilidade de água no afloramento rochoso seriam devidas, entre outras causas, ao tamanho das ilhas de solo onde D. tuberose ocorre e as características de declividade e orientação da vertente da superfície rochosa onde a ilha esta instalada. A partir das observações no ambiente natural, pergunta-se se os padrões de assimilação em D. tuberose relacionados ao CAM apresenta variações associadas as características das ilhas de solo onde ocorre. Para investigar esta relação, plantas de D. tuberose foram estudadas em campo e em condições semi-controladas em casa de vegetação. A técnica utilizada para caracterização dos padrões de CAM foi a de titulação da acidez do mesofilo. Tanto em grupos experimentais submetidos a períodos distintos de suspensão da rega como em amostras obtidas diretamente do campo, observam-se padrões de variação da acidez que podem ser associados a variações no suprimento hídrico. As variações foram caracterizadas através da diferença na acidez entre o inicio da manha e o final da tarde. A redução na amplitude da variação coincidiu com a intensidade da restrição no suprimento hídrico. Entretanto, a variação foi associada a redução do valor da acidez no final do dia, o que estaria associado a uma redução na recaptura do CO2 durante o período de iluminação. A maior influencia detectada nas plantas em campo foi a umidade relativa do ar. Conjectura-se um papel da assimilação de água através das folhas de D. tuberose como um atributo essencial a ocupação do substrato rochoso na Pedra Grande - Atibaia / Plants with crassulacean acid metabolism are able to support conditions of low availability of water through a carbon dioxide concentration system which increases the efficiency of use of water through an overnight CO2 fixture under low deficit of vapor pressure between the leaf and the atmosphere. This process favors the occupation of arid regions and places where water can quickly become unavailable. The occurrence of this type photosynthetic covers a wide range of taxa, including the Bromeliaceae family. Among the species of Bromeliaceae presenting CAM is Dickya tuberose, a species that has a great abundance on rocky outcrops associated with natural monument of Pedra Grande - Atibaia - SP. At this site, D. tuberose has a role in vegetation associated with surface rock exposed as an essential component of communities called \"vegetation islands\". The conditions prevailing in the rocky outcrop of Pedra Grande are of great scarcity of land and water, and extreme exposure. The variations in water availability in rocky outcrop would be due, among other reasons, the size of the islands where vegetation D. tuberose occurs and the characteristics of inclination and orientation of the slope of the rocky surface where the island is installed. From the observations in the natural environment, wonders whether the patterns of assimilation in D. tuberose related to CAM presents variations associated characteristics of vegetation islands where it occurs. To investigate this relationship, plants of D. tuberose were studied in field and semi-controlled conditions in a greenhouse. The technique used to characterize patterns of CAM was the titration acidity of the mesophyll. In both experimental groups undergoing different periods of suspension of irrigation as in samples obtained directly from the field, there are patterns of variation in acidity that may be associated with variations in water supply. The changes were characterized by the difference in acidity between the early morning and late afternoon. The reduction in amplitude of the variation coincided with the intensity of the restriction in water supply. However, the variation was associated with reduced acidity value at the end of the day, which would be associated with a reduction in the recapture of CO2 during the illumination period. The major influence detected on plants in the field was the relative humidity. It is conjectured a part of the assimilation of water through the leaves of D. tuberose as an essential attribute of the occupation of substrate in Pedra Grande - Atibaia

Orientador: Tatiane Maria Rodrigues / Resumo: Em espécies perenes de ambientes sazonais, o estabelecimento de um sistema radicular profundo é uma das estratégias de adaptação dessas espécies ao estresse hídrico. Nessas plantas, é bem estabelecido que a maior parte da absorção de água e íons ocorre em raízes finas não-suberizadas e que as porções mais jovens das raízes são as mais ativas nesse processo. Uma vez que modelos de mudança climática preveem uma redução de chuvas de até 20% entre a primeira e a última década do século XXI em grande faixa do globo terrestre, incluindo o Brasil, entender o efeito da redução da precipitação sobre a anatomia das raízes faz-se cada vez mais importante. O objetivo desse trabalho foi estudar a anatomia de raízes de Eucalyptus grandis em diferentes profundidades do solo e investigar o efeito da limitação da precipitação nos aspectos estruturais dessas raízes. Um delineamento experimental foi estabelecido em junho de 2010 com um clone de E. grandis em uma plantação da Estação Experimental de Ciências Florestais de Itatinga (EECFI), SP, Brasil. Dois tratamentos (dois regimes de água) foram aplicados em blocos separados de 2010 a 2014: tratamento TO (controle) sem a intercepção de chuvas e tratamento TR com interceptação de 37% das chuvas por lâminas de plástico transparente. Em 2014, trincheiras de até 17 metros de profundidade foram abertas no solo, sendo uma para cada tratamento. Cada trincheira foi centralizada entre quatro indivíduos de E. grandis. Raízes finas laterais foram coletada... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In perennial species the establishment of a deep root system is an adaptive strategy to water deficit. In these plants it is well established that most of the absorption of water and ions in perennial woody species occurs in fine non-suberized roots and that younger portions of roots are the most active in this process. Since models of climate changes predict a rainfall reduction in the next years of 20% until the first and the last decade of 21st century in global large scale, including Brazil, to understand the effect of rainfall reduction on the anatomy of these roots becomes even more important. This study aims to study the anatomy of Eucalyptus grandis roots at different soil depths investigating the effect of limiting the rainfall in structural aspects of these roots. An experiment was set up in June 2010 with a clone of Eucalyptus grandis used in commercial plantations by the Suzano Company in Estação Experimental de Ciências Florestais de Itatinga (EECFI), SP, Brazil. Two treatments (two water systems) were applied in separated blocks from 2010 to 2014: treatment TO (control) without rainfall interception and treatment TR (reduction) with 37% of rainfall interception by transparent plastic sheeting panels. In 2014, trenches of until 17 m of depth were opened in the soil, one for each treatment respectively. Each trench was centered between fours individuals of E. grandis. Fine lateral roots with were collected at 0-50cm and 12-16m of depth and separated in four catego... (Complete abstract click electronic access below) / Mestre

O Brasil é o segundo maior produtor de feijão, sendo a espécie mais cultivada o Phaseolus vulgaris L. Entre as três possíveis safras exploradas no Brasil, aquela que gera a maior produção é a da seca. Por outro lado, como a maioria das lavouras emprega pouca tecnologia, um dos problemas desta cultura é o estresse hídrico, que leva a uma redução na produtividade. Dessa forma, a identificação de genes que controlam os mecanismos de defesa e adaptação do feijoeiro à falta de água seria de grande utilidade. Nos últimos anos, muitas informações ômicas do feijoeiro foram geradas, criando uma visão integrada deste organismo e oferecendo uma complexa rede de interações entre genes e seus produtos. Este trabalho teve como objetivo central à identificação de genes diferencialmente expressos no sistema radicular de um genótipo de feijoeiro resistente ao estresse hídrico (BAT 477), quando submetido a uma interrupção de irrigação durante seu desenvolvimento. Foi construída uma biblioteca subtrativa de cDNA (SSH), que representou os genes diferencialmente expressos no genótipo resistente, utilizando-se como driver o genótipo Carioca 80SH (suscetível a seca). Foram obtidos 1572 reads válidos, sendo 931 destes singletons e 189 contigs com uma média de seis reads por cluster. A anotação das sequências foi conduzida via BLASTX, sendo consideradas para anotação somente os melhores resultados dos produtos gênicos similares com E- Value \'<OU=\' 10-5. A classificação funcional foi feita tendo-se como base modelos descritos para plantas (modelo CS e MIPS) e os resultados foram agrupados em seis classes funcionais distintas. As análises de bioinformática ajudaram na identificação de genes descritos como envolvidos na resposta da planta ao estresse hídrico. Entre eles: proteínas do grupo LEA; fatores de transcrição como DREB, NAC e proteínas ricas em leucina; enzimas sintetizadoras de carboidratos incluindo trehalose, sacarose e rhamnose; proteínas ricas em prolina; receptores de hormônios (ABA, etileno); aquaporinas; chaperonas; ubiquitinas; nodulinas; e proteínas associadas à fotossíntese e à respiração. A fim de se obter a validação das ESTs anotadas, foi conduzido um experimento de PCR em tempo real confrontando os padrões de expressão de 15 genes sob quatro tratamentos: ambos os genótipos sob estresse e respectivos controles. Três replicatas biológicas foram adotadas e dois genes de referência (act e skip2) foram escolhidos para normalização interna dos dados. Os padrões de expressão gênica obtidos confirmam a hipótese de que tais genes são mesmo mais expressos no genótipo resistente, embora não sejam exclusivos já que uma quantidade menor de tais transcritos também foi detectada no genótipo suscetível / Brazil is the second biggest producer of common bean, being Phaseolus vulgaris L. the most cultivated species. Among the three possible harvests exploited in Brazil, drought is the one which generates the greatest production. On the other hand, as the majority of the households employees low technology, one of the problems of this culture is drought stress that leads to a reduction in the productivity. So, the identification of gene that controls the mechanisms of defense and adaptation of common bean to the lack of water would be very useful. In the past years, many omics information of common bean have been generated, creating an integrated view of this organism and providing a complex network between genes and its products. The main goal of this work was the identification of differentially expressed genes in a genotype of common bean resistant to drought stress (BAT 477), when submitted to a interruption of irrigation during its development. It was build a cDNA suppression subtractive hybridization library (SSH), which represented the differentially expressed genes, on the resistant genotype, having as driver the genotype Carioca 80SH (susceptible to drought). It was obtained 1572 valid reads, being 931 singletons and 189 contigs, with the average of 6 reads per cluster. The sequences annotation was conducted via BLAST X, considering only the best similarity results with E value \'<OU=\' 10-5. The functional classification was done adopting models described for plants (CS and MIPS) and the results were grouped into six different functional classes. Bioinformatic analyses contribuited to the identification of genes described as involved on plants response to drought stress. Among them: LEA proteins; transcription factors like DREB, NAC and leucine-rich proteins; carbohydrates synthesizers enzymes like the ones for trehalose, sucrose and rhamnose; proline-rich proteins; hormone receptors (ABA and ethylene); aquaporins; chaperones; ubiquitins; nodulins; and proteins associated with photosynthesis and respiration. In order to validate the ESTs annotated, a RT-qPCR experiment was conducted comparing the expression patterns of 15 genes under four treatments: both genotypes under stress and their respective controls. Three technical replicates were used and two reference genes (act and skip2) were chosen for intern data normalization. The gene expression patterns obtained confirm the hypothesis that such genes are more expressed on the resistant genotype although they are not exclusive since a lower levels of these transcripts were also detected in the susceptible genotype