Orientador: Maria Inês Tiraboschi Ferro / Coorientador: Miguel Angelo Mutton / Banca: Luciana Rossini Pinto / Banca: André Luiz Julien Ferraz / Banca: Rinaldo Cesar de Paula / Banca: Samira Domingues Carlin / Resumo: A cana-de-açúcar tem adquirido crescente importância no cenário mundial, não somente pela produção do açúcar, mas devido ao grande potencial como cultura bioenergética, sendo utilizada na produção do etanol, uma fonte energética alternativa, ecologicamente limpa e renovável e como co-geradora de energia elétrica através da queima do bagaço. O Brasil destaca-se mundialmente como o maior produtor da cultura e exportador de açúcar e etanol. Porém, esta cultura está sujeita a diferentes estresses ambientais, dentre os quais, a disponibilidade de água representa o principal fator que influencia o crescimento e o desenvolvimento vegetal e, conseqüentemente, a produtividade. Dessa forma, para a identificação dos possíveis genes envolvidos nas respostas de tolerância à seca em cana-de-açúcar, realizou-se, por meio da técnica de cDNA-AFLP, a análise comparativa do perfil de transcritos de duas cultivares tolerantes, SP83-5073 e RB867515 com o perfil de expressão da cultivar sensível à seca, SP86-155, quando submetidas a suspensão da rega por 1, 3, 5 e 10 dias. Com 14 combinações de primers seletivos EcoRI/MseI, foram detectados no gel de poliacrilamida, 1.316 fragmentos derivados de transcritos (FDTs) que foram diferencialmente expressos entre as cultivares tolerantes e a sensível, submetidas ao estresse hídrico (1, 3, 5 e 10 dias). Destes, 630 FDTs foram identificados na cultivar SP83-5073 e 686 FDTs na cultivar RB867515. A análise comparativa do perfil de expressão associado ao genótipo da cana-de-açúcar revelou fragmentos de transcritos similares a genes envolvidos em eventos moleculares relacionados às respostas das plantas a diferentes estresses, inclusive ao causado pela seca. Estes genes atuam na sinalização hormonal, regulação da transcrição gênica, senescência, transposição de DNA... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The sugarcane has increased importance in the world not only for sugar production, but also due to the great potential as a generator energy because ethanol production, it is a renewable, alternative energy source, ecologically clean and as energy cogeneration through burning of bagasse. Brazil is the world's largest crop producer and exporter of sugar and ethanol. However, this crop is exposed to different kinds of stress, among which; the availability of water is noticeable as the main factor that negatively influences the vegetable growth and, therefore, the productivity. In order to identify the possible genes involved in the responses to drought tolerance in sugarcane crops, the comparative analysis of transcripts profile from two tolerant cultivars, SP83-5073, RB867515 and the sensitive cultivar SP86-5073, after has been submitted to the interruption of irrigation for 1, 3, 5 and 10 days were performed using the cDNA-AFLP technique. With 14 combinations of EcoRI/MseI selective primers, there was detected on a polyacrylamide gel, 1,316 transcript-derived fragments (TDFs) differently expressed between tolerant and sensitive cultivars submitted to water deficit (1, 3, 5 and 10 days). From these, 630 TDFs were identified on the cultivar SP83-5073 and 686 TDFs corresponded to the cultivar RB867515. The comparative analysis of genotype dependent gene expression profile in sugarcane revealed fragments similarity with genes involved in many molecular events related to the responses of plants to different stresses, including those caused by drought. These genes work in hormonal signaling, regulation of gene transcription, senescence, DNA transposition, transport of amino acids, respiration and metabolism of carbohydrates. However, nine TDFs possibly represent still unknown genes, which may be developing important roles on the responses to drought and contributing to the trait of tolerance / Doutor

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No. of bitstreams: 1 PDF - Rayssa Ribeiro da Costa.pdf: 1490585 bytes, checksum: e8add9fe6f4f56eb0481ceb023a6dec1 (MD5) Previous issue date: 2016-02-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cowpea is a one of the legume most consumed in the North and Northeast of Brazil, being a major component of the population diet. In the critical stages, the severity of drought is one of the factors that limit the plant growth. However studies have reported that effects of drought on the plant can be mitigated through the application of endogenous elicitors such as salicylic acid (SA). Thus, the objective of this work was to evaluate cowpea genotypes subjected to drought with salicylic acid applications. The research consisted of a 2x2x5 factorial, two cowpea genotypes (BRS Paraguaçu and Pingo de Ouro 1-2), two irrigation levels (one befitting to soil moisture near field capacity, 10 and 15 kPa, and another in a condition of stress (60 to 80kPa) and five concentrations of salicylic acid (zero; 0.1; 0.5; 1.0; 2.0 mM) applied 15 days after sowing each three days during six days. The combination of these factors resulted in 20 treatments, arranged in a completely randomized design with three replications, and the experimental plot consisted of 3 working vessels initially containing 6 plants. Growth evaluations ( plant height, leaf number, stem diameter and dry mass of shoots), physiological assessments (photosynthesis, stomatal conductance, transpiration, leaf water potential and intrinsic efficiency in water use), biochemical (activities of peroxidase ascorbate - APX, catalase - CAT, superoxide dismutase - SOD and free proline content) and production (long pods, weight of pods, number of grains per pod, grain diameter, 100 seed weight, moisture content and productivity) were analyzed. The application of 1.0 mM SA provided benefits to physiological parameters in both genotypes at both conditions. The activity of antioxidant enzymes increases in water drought conditions, but no responded to foliar application of salicylic acid. Although not significant, the proline accumulation in drought conditions increased the tolerance in Pingo Gold-1-2. Pingo de Ouro 1-2 had lower productivity, but increased tolerance to drought. In the vegetative phase, the foliar application of salicylic acid attenuates water deficit in cowpea. tolerância ao déficit. Pingo de Ouro-1-2 obteve menor produtividade, porém uma maior tolerância ao déficit hídrico. A aplicação foliar do ácido salicílico na fase vegetativa atenua o déficit hídrico moderado em feijão-caupi. / O feijão-caupi é uma das leguminosas mais consumidas no Norte e Nordeste do Brasil, sendo um dos principais componentes da dieta alimentar da população. A severidade do déficit hídrico nas fases críticas da cultura é um dos fatores que limitam a cultura, entretanto estudos relatam que os efeitos do déficit hídrico na planta podem ser mitigados através da aplicação de eliciadores endógenos como o ácido salicílico (AS). Dessa forma, objetiva-se com este trabalho, avaliar genótipos de feijão-caupi submetidos a estresse hídrico e aplicações de ácido salicílico. A pesquisa constou de um fatorial de 2x2x5, sendo dois genótipos de feijão-caupi (BRS Paraguaçu e Pingo de Ouro-1-2), duas lâminas de irrigação (uma condizendo-se a umidade do solo próximo à capacidade de campo, entre 10 e 15 kPa, e outra numa condição de estresse (entre 60 e 80kPa); e cinco concentrações de ácido salicílico (zero; 0,1; 0,5; 1,0; 2,0 mM) aplicadas 15 dias após a semeadura em intervalos de três dias, durante seis dias. A combinação dos três fatores resultou em 20 tratamentos, arranjados no delineamento experimental inteiramente casualizado, com três repetições e a parcela experimental composta de 3 vasos úteis, contendo inicialmente 6 plantas. Foram realizadas avaliações de crescimento (altura da planta, número de folhas e massa seca da parte aérea), avaliações fisiológicas (fotossíntese, condutância estomática, transpiração, potencial hídrico foliar e eficiência intrínseca no uso de água), bioquímicas (atividades da ascorbato peroxidade - APX, catalase - CAT, superóxido dismutase – SOD e teor de prolina livre) e de produção (comprimento de vagens, peso das vagens, número de grãos por vagem, diâmetro do grão, peso de 100 sementes, teor de umidade e produtividade). A aplicação de ácido salicílico a 1,0 mM propiciou benefícios aos parâmetros fisiológicos em ambos os genótipos nas duas condições hídricas. A atividade das enzimas antioxidantes aumenta em condições de déficit hídrico, porém sem resposta a aplicação foliar de ácido salicílico. O acúmulo de prolina em condições de déficit hídrico, mesmo não sendo significativo, confere ao Pingo de Ouro-1-2 maior

A vegetação ao longo do rio Amazonas é submetida a dois períodos muito bem marcados ao longo do ano: estação de cheia e estação seca. Características morfológicas e fisiológicas das espécies de várzea foram amplamente descritas, porém, uma das principais características é o metabolismo de carboidratos que tem sido muito pouco explorada. Com o objetivo de estudar a resiliência dos carboidratos não estruturais da árvore amazônica Senna reticulata (Leguminosae), a mesma foi cultivada sob diferentes estresses abióticos, como luz baixa combinada com elevada concentração de CO2 atmosférico, bem como disponibilidade hídricas distintas (alagamento das raízes e seca). Os resultados encontrados reforçam o papel do amido nas folhas como principal órgão armazenador desta espécie e também a alta resiliência desta reserva, que foi mantida mesmo em condições de baixa irradiância, em alagamento e em condições de seca. Estes resultados também destacam a forte ligação entre as reservas de amido e a manutenção do crescimento. Além disso, esta espécie mostrou estratégias ecofisiológicas distintas em relação a diferentes estresses hídricos (alagamento x seca) e uma alta capacidade de se recuperar depois de um período de seca, padrão este que pode estar diretamente relacionado à manutenção do crescimento durante o estabelecimento de plantas jovens no primeiro período de fase terrestre / The vegetation along the Amazon River is subjected to two markedly periods: flooded and drought seasons. Morphological and physiological traits for floodplain species have been largely described; however, one of the main features is the carbohydrate metabolism, which has been poorly explored. In order to study the resilience of non-structural carbohydrates of the Amazonian tree Senna reticulate (Leguminosae), this species was grown under different abiotic stresses such as low light combined with elevated CO2 and distinct water availabilities (waterlogging and drought). Results reinforce the role of starch in leaves as a main storage organ of this species and also the high resilience of this reserve under low light, waterlogging and drought conditions. These findings also highlight the tight connection between starch reserves and growth maintenance. Additionally, this species has shown very distinct eco physiological strategies to cope with different water availabilities and a high ability to recover after drought that might be strictly related to growth maintenance during seedling establishment in the first period of terrestrial phase

Abscisic acid (ABA) induces RD22, responsive to dehydration stress gene. Salicylic acid (SA) has been the focus of research due to its role against pathogens and abiotic stress. Interaction between ABA and SA signaling pathways is still poorly understood. SA-Binding Protein 2 (SABP2) converts methyl salicylate to SA. An attempt was made to identify proteins that interact with SABP2 using a yeast 2-hybrid screening. Several interactors were identified. One of them, SA-Binding Protein 2 Interacting Protein-355 (SBIP-355), showed high homology to RD22. Bioinformatic approaches showed that SBIP-355 contains a BURP domain. Phylogenetic analysis reveals that SBIP-355 clustered into the clade of RD22-like proteins. Thus, SBIP-355 gene might be a stress-inducible gene and encodes a dehydration-responsive protein, which is important for the stress tolerance of tobacco. The complementary DNA (cDNA) of tobacco SBIP-355 was cloned into pDEST-17 vector and then expressed in E. coli to detect the expression of SBIP-355 protein.

Plants inherently produce polyphenols (i.e., antioxidants) as a response to reduce oxidative stress caused by abusive environmental pre- and postharvest conditions. These antioxidants, as well as vitamin C, are present in considerable levels in strawberries; however, excessive oxidative stress brought on by improper postharvest handling conditions can reduce the levels of antioxidants in the fruit and shorten the shelf-life of strawberries. Nevertheless, it may be possible to utilize strawberry’s naturally occurring polyphenols to reduce postharvest stress and extend their shelf life. The polyphenolic profile has been previously investigated in several strawberry cultivars, however no studies have determined the unique polyphenolic profiles of three important Florida strawberry cultivars (‘Florida Radiance’, Sweet Sensation® ‘Florida 127’and ‘Florida Beauty’) at harvest and during cold storage. Therefore, in order to better understand the distribution of individual polyphenols within these cultivars and their impact on postharvest shelf-life, this study examined the polyphenolic profiles throughout 7 days of cold storage (1 °C) using an HPLC-DAD. The activity of phenylalanine ammonia lyase (PAL), a key enzyme in the biosynthesis of polyphenols, and polyphenol oxidase (PPO), the enzyme responsible for polyphenol degradation, were also examined during cold storage to understand their possible influences on postharvest synthesis or degradation of polyphenols. This study revealed that the polyphenolic profile of strawberry fruit was genotype dependent; however, pelargonidin 3-glucoside was consistently the anthocyanin found in higher concentrations in the fruit regardless of the cultivar. Apart from the anthocyanins, the flavonols showed the most variation among the three cultivars. PAL was slightly induced during strawberry postharvest storage suggesting that a stress response occurred during cold storage while PPO showed variable induction patterns across all three cultivars most likely due to their different polyphenol profiles. Analysis of the distribution of polyphenols in the cortex and pith of strawberries showed that polyphenols were mostly concentrated in the cortex of the fruit and that the concentration of individual polyphenol in each fruit tissue varied by cultivar. These results indicate that the oxidative stress response varies in each of the strawberry cultivars studied contributing to their unique polyphenolic profile. Results from this study can ultimately help to identify the polyphenols and enzymes related to superior postharvest quality in future studies.

<p>Excess excitation energy, mechanical injury and defence against pathogens, each trigger rapid production of reactive oxygen species (ROS) in <i>Arabidopsis thaliana</i> leaves. ROS, such as hydrogen peroxide (H₂O₂), are required for the induction of systemic acquired acclimation and may lead to redox changes in photosynthetic electron transport (PET). On one hand, enhanced ROS production during stress can destroy cells, and on the other, ROS can also act as signals for the activation of stress responsive and defensive pathways.</p><p>In this work, physiological and molecular analyses of <i>Arabidopsis</i> mutants and transgenic lines were applied to investigate the signalling network controlling biotic and abiotic stress responses. A key enzyme of the antioxidant network is encoded by <i>ASCORBATE PEROXIDASE 2 (APX2</i>). Wounded leaves showed low induction of<i> APX2 </i>expression and when exposed to excess light, <i>APX2</i> expression was increased synergistically. Signalling pathways dependent upon jasmonic acid, chitosan and abscisic acid were not involved in the wound-induced expression of <i>APX2</i>, but PET was required, and APX2 induction was preceded by a depressed rate of CO₂fixation.</p><p>Analysis of<i> lsd1</i> (<i>LESION SIMULATING DISEASE 1</i>) strongly suggests that light acclimatory processes and pathogen defences are genetically and functionally linked. It is important to know that LSD1 type of mutants have mainly been studied with regard to pathogenesis. From this work, it reveals that association of LSD1 with hypersensitive response may only be supplementary.</p><p><i>GLUTATHIONE PEROXIDASES</i> <i>(GPXs)</i> are another major family of ROS scavenging enzymes. Analysis of the <i>Arabidopsis </i>genome database revealed a new open-reading frame, thus increasing the total number of <i>AtGPX </i>gene family to eight (<i>AtGPX1-AtGPX8</i>). <i>Arabidopsis thaliana</i> transgenic lines with reduced expression of both putative chloroplastic isoforms (<i>AtGPX1 </i>and <i>AtGPX7</i>) and <i>AtGPX7</i> knock-out mutant (ko-<i>GPX7</i>) were more sensitive to photo-oxidative stress but had a reduced bacterial growth rate when inoculated with virulent strains <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 and<i> P.s.t. maculicola</i> strain ES4326, indicating increased resistance to pathogenesis. This, to our knowledge, is the first functional and genetic analysis of chloroplastic GPXs in plants, and confirms that light and chloroplastic ROS metabolism is important for basal resistance against virulent pathogens.</p><p>The above results confirm that light sensing, light acclimatory processes and photo-produced ROS also govern pathogen defence pathways. This has a great ecological relevance for Darwinian fitness of plants growing in the natural environment, where simultaneous pathogen attack and fluctuations in light, temperature and other environmental factors make rapid acclimation a constant necessity. Molecular, biochemical and physiological analysis of pathogen responses in mutants impaired in light sensing, EEE-dissipatory mechanisms, and similar analysis of light acclimatory processes in mutants impaired in pathogen defences may prove to be seminal.</p>

Excess excitation energy, mechanical injury and defence against pathogens, each trigger rapid production of reactive oxygen species (ROS) in Arabidopsis thaliana leaves. ROS, such as hydrogen peroxide (H2O2), are required for the induction of systemic acquired acclimation and may lead to redox changes in photosynthetic electron transport (PET). On one hand, enhanced ROS production during stress can destroy cells, and on the other, ROS can also act as signals for the activation of stress responsive and defensive pathways. In this work, physiological and molecular analyses of Arabidopsis mutants and transgenic lines were applied to investigate the signalling network controlling biotic and abiotic stress responses. A key enzyme of the antioxidant network is encoded by ASCORBATE PEROXIDASE 2 (APX2). Wounded leaves showed low induction of APX2 expression and when exposed to excess light, APX2 expression was increased synergistically. Signalling pathways dependent upon jasmonic acid, chitosan and abscisic acid were not involved in the wound-induced expression of APX2, but PET was required, and APX2 induction was preceded by a depressed rate of CO2 fixation. Analysis of lsd1 (LESION SIMULATING DISEASE 1) strongly suggests that light acclimatory processes and pathogen defences are genetically and functionally linked. It is important to know that LSD1 type of mutants have mainly been studied with regard to pathogenesis. From this work, it reveals that association of LSD1 with hypersensitive response may only be supplementary. GLUTATHIONE PEROXIDASES (GPXs) are another major family of ROS scavenging enzymes. Analysis of the Arabidopsis genome database revealed a new open-reading frame, thus increasing the total number of AtGPX gene family to eight (AtGPX1-AtGPX8). Arabidopsis thaliana transgenic lines with reduced expression of both putative chloroplastic isoforms (AtGPX1 and AtGPX7) and AtGPX7 knock-out mutant (ko-GPX7) were more sensitive to photo-oxidative stress but had a reduced bacterial growth rate when inoculated with virulent strains Pseudomonas syringae pv. tomato DC3000 and P.s.t. maculicola strain ES4326, indicating increased resistance to pathogenesis. This, to our knowledge, is the first functional and genetic analysis of chloroplastic GPXs in plants, and confirms that light and chloroplastic ROS metabolism is important for basal resistance against virulent pathogens. The above results confirm that light sensing, light acclimatory processes and photo-produced ROS also govern pathogen defence pathways. This has a great ecological relevance for Darwinian fitness of plants growing in the natural environment, where simultaneous pathogen attack and fluctuations in light, temperature and other environmental factors make rapid acclimation a constant necessity. Molecular, biochemical and physiological analysis of pathogen responses in mutants impaired in light sensing, EEE-dissipatory mechanisms, and similar analysis of light acclimatory processes in mutants impaired in pathogen defences may prove to be seminal.

During the water-to-land transition, that occurred approximately 450 MYA, novel habitats wererevealed to the emerging plants. This terrestrial habitat was a harsh environment compared to theaquatic, with shifting substrate content, irregular supply of water, damaging UV-radiation andrapid fluctuating temperatures. Non-specific lipid transfer proteins (nsLTP) are today only foundin the land living plants and not in the green algae. This suggests that these genes might haveevolved to help the plants cope with the stressful conditions. In this study the expression patternhas been analysed of the nsLTPs in the moss Physcomitrella patens during the possible conditionsthat raised during the water-to-land transition. The moss was exposed to salt, UV-B, drought, copper, cold and osmotic stress. Quantitative real-time PCR was used to analyse the transcription levels. I found that six genes were upregulated during either cold, dehydration or UV-B stress. This suggest that these genes are involved in the plant defense against these abiotic stresse

ABSTRACT TRANSFORMATION OF POTATO WITH MYB4 TRANSCRIPTION FACTOR AND EVALUATION OF ABIOTIC STRESS TOLERANCE AND GENE EXPRESSION PROFILES IN TRANSGENIC PLANTS Kalemtas, G&uuml / ls&uuml / m Ph.D., Department of Biology Supervisor: Prof. Dr. H&uuml / seyin Avni &Ouml / ktem February 2011, 257 pages Potato (Solanum tuberosum L. cv. Kennebec) was transformed via Agrobacterium tumefaciens (EHA105) harbouring two different binary vectors containing Oryza sativa myb4 gene, which encodes MYB4 transcription factor / under the control of CaMV35S promoter or cold inducible COR15a promoter. The transgenic plants were not growth retarded and there was no significant difference (p&lt / 0.05) in their tuber yield compared to wild-type plants. Wild-type and transgenic plants were subjected to abiotic stresses to compare their stress tolerances. There was no significant difference in boron, freezing and drought tolerances of wild-type and transgenic lines. Two of the transgenic lines were more salt tolerant than wild-type with respect to growth parameters. Transcriptomes of wild-type and these two lines, one expressing myb4 under the control of 35S promoter and the other COR15a promoter, were analyzed to elucidate the myb4-regulated processes and downstream target genes in potato. Differentially regulated genes in transgenic lines showed that myb4 controls a large and complex transcriptional network associated with diverse cellular processes, primarily defense and rescue, metabolism and development. Genes involved in sucrose synthesis, some peroxidases and CBF3 transcription factor were up-regulated in transgenic plants upon exposure to freezing stress. This suggested that myb4 may configure freezing response in potato primarily by oxidative stress defence mechanisms, osmotic adjustment or activation of CBF3 regulated genes that may confer cold tolerance. Despite up-regulation of these stress related genes, transgenic potato was not more drought or freezing tolerant compared to WT under the tested conditions. Further experiments should be conducted to better elucidate the involvement of these genes in regulation of stress response in transgenic potato expressing myb4.

Wheat is the most important grain crop grown in our country providing greatest part of the daily nutritional requirement. Abiotic factors including salinity, drought, cold and heat stresses affect quality and yield of wheat varieties used for the production of both bread and pasta flour. NAC proteins form one of the widest families of plant specific transcription factors. Members of this family are related with development, defense and abiotic stress responses. TaNAC69-1 and TtNAM-B2 genes were isolated from T.aestivum and T.turgidum, respectively. Then they were cloned into different monocot and dicot expression vectors to be used for further wheat and tobacco genetic transformation studies. To understand effects of salinity, drought, cold and heat stresses on expression profiles of TaNAC69-1 and TtNAM-B2 genes, quantitative real time PCR was performed. The time series expression profiles of TaNAC69-1 show that it was signi