Submitted by LUANA JANDHY MANTOVANINI (lumantovanini@gmail.com) on 2017-08-29T19:11:30Z No. of bitstreams: 1 Dissertação_Luana_Jandhy_Mantovanini.pdf: 1646514 bytes, checksum: 4c536d5f2d4ac50ce5a852c9b10ce27c (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-08-29T19:18:01Z (GMT) No. of bitstreams: 1 mantovanini_lj_me_jabo.pdf: 1646514 bytes, checksum: 4c536d5f2d4ac50ce5a852c9b10ce27c (MD5) / Made available in DSpace on 2017-08-29T19:18:01Z (GMT). No. of bitstreams: 1 mantovanini_lj_me_jabo.pdf: 1646514 bytes, checksum: 4c536d5f2d4ac50ce5a852c9b10ce27c (MD5) Previous issue date: 2017-05-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A cana-de-açúcar é atualmente uma das principais culturas da agroindústria mundial. Devido à ampla expansão de seu plantio é submetida constantemente a solos não produtivos. A presença de moléculas tóxicas no solo, como o alumínio (Al3+), interfere diretamente no desenvolvimento radicular ocasionando baixa absorção de água e nutrientes levando a pouca produtividade e desenvolvimento das plantas. Os microRNAs tem sido descritos como um dos fatores responsáveis pela regulação gênica e a descoberta dessas moléculas abre um novo caminho para a elucidação da tolerância e adaptação das plantas aos estresses abióticos. Este estudo visou avaliar em duas cultivares de cana-de-açúcar (CTC-2 e RB855453) a expressão dos microRNAs miR159, miR164 e miR168, associados à resposta ao alumínio em espécies como Arabidopsis thaliana, arroz (Oriza sativa) e tabaco (Nicotiana tabacum). Algumas características foram avaliadas, como densidade de raízes (DS), área foliar (AR), produção de massa seca (MS) e teor de prolina nas folhas, em quatro cultivares de cana-de-açúcar submetidas a diferentes concentrações de alumínio. A cultivar CTC 2 foi classificada como tolerante e a RB855453 como sensível ao estresse. Ambas foram selecionadas e em sistema de hidroponia submetidas novamente ao estresse pela toxidez de alumínio na concentração de 221 μmol L -1 . Parâmetros fisiológicos foram mensurados (área foliar, potencial osmótico, taxa de fotossíntese, transpiração, condutância estomática) e a expressão dos miRNAs 159, 164 e 168 avaliada por PCR em tempo real. Plantas das duas cultivares apresentaram alterações fisiológicas e morfológicas ao longo do estresse, com redução significativa para a área foliar da cultivar RB855453. O miR164 e 159 foram induzidos nas duas cultivares, principalmente após 72 horas de estresse, e o miR168 diferencialmente expresso. Esses miRNAs regulam genes e fatores de transcrição que estão envolvidos na resposta e desenvolvimento da planta diante ao estresse por alumínio. / The sugarcane is currently one of the main crops of global agribusiness. Due to the wide expansion of its plantation is constantly subjected unproductive soils. The presence of toxic molecules in the soil, such as aluminum (Al3+), directly affects root development, leading to poor absorption of nutrients and water leading to low productivity and development of plants. Studies of the interactions of plants with the environment are being conducted to clarify the resistance or susceptibility of various cultures, favoring the discovery of important mechanisms that participate in physiological and molecular responses to environmental stresses. MicroRNAs have been described as one of the factors responsible for gene regulation and the discovery of these molecules opens a new path for the elucidation of tolerance and adaptation of plants to abiotic stresses. This study evaluated in two sugarcane varieties the expression of microRNA miR159, miR164 and miR168, associated with the response to the aluminum species such as Arabidopsis thaliana, rice (Oryza sativa) and tobacco (Nicotiana tabacum). Some characteristics were evaluated, such as density (DS), area (AR), dry mass production (DM) and proline content, in four sugarcane cultivars submitted to different concentrations of aluminum. CTC 2 cultivar was classified as tolerant and RB855453 as stress sensitive. Both were selected and in a hydroponics system again submitted to stress by the aluminum toxicity in the concentration of 221 μmol L-1. Physiological parameters were measured (leaf area, osmotic potential, photosynthesis rate, transpiration, stomatal conductance) and the expression of miRNAs 159, 164 and 168 evaluated by real-time PCR. Plants of both cultivars presented physiological and morphological changes along the stress, with a significant reduction for the leaf area of cultivar RB855453. The miR164 and 159 were induced in the two cultivars, mainly after 72 hours of stress, and the miR168 differentially expressed. These miRNAs regulate genes and transcription factors that are involved in the response and development of the plant in the face of aluminum stress.

Barley and wheat are the two most important crop species in Turkey. Molecular studies for increasing crop yield of these species are very important for the economic benefits of Turkey. Powdery mildew and yellow rust are the two main pathogens, infecting barley and wheat, respectively in our country and causing a great amount of yield loss each year. Till now, classical genetics studies were performed in order to develop resistant barley and wheat cultivars, but these studies have not been succesful. Therefore, molecular plant-pathogen interactions studies are starting to become the new tool to fight against pathogens. In this thesis, two important aspects of plant microbe interactions were investigated. In the first part, the role of microRNAs (miRNAs) in powdery mildew-barley pathosysytem, and yellow rust-wheat pathosystem were studied. The expression levels of miRNAs and their putative targets were investigated via miRNA microarray analysis and qRT-PCR, respectively, in response to virulent and avirulent pathogen infections. These data were used to establish a new model for powdery mildew-barley and yellow rust-wheat pathosystems. In the second part, functional analysis of a novel F-box gene, which was a ZTL-type F-box, was performed by using Barley Stripe Mosaic Virus mediated Virus Induced Gene Silencing. This F-box gene (HvDRF) (Hordeum vulgare Disease Related F-box) was induced upon yellow rust infection and we studied its role in powdery mildew infection. The results confirmed HvDRF as a positive regulator of race specific immunity and enlarged the roles of ZTL-type F-box proteins to biotic stress responses.

Barley is one of the most important crop species in Turkey and powdery mildew is one of the most common pathogen decreasing yield in barley. For this problem, agricultural biologists apply breeding technologies in order to select and propagate resistant barley cultivars. However, this is not a permanent solution since pathogens evolve rapidly to overcome plant resistance mechanisms. On the other hand, molecular plant pathologists are trying to understand basic mechanisms underlying plant-pathogen interactions by using molecular tools in order to develop long term solutions for preventing yield loss. In this thesis, miR159 mediated regulation of barley GAMyb transcription factor is studied. According to microRNA microarray results regarding to infection with powdery mildew pathogen Blumeria graminis f.spp hordei (Bgh) at different time points, miR159 expression level showed significant differences. Bioinformatics analysis revealed that miRNA159 targets GAMyb gene in barley. In order to investigate this relationsh&amp / #8223 / p, both miRNA and miRNA target were cloned into GFP containing expression vectors through Gateway cloning and resulting vectors were transformed into Nicotiana benthamiana through Agrobacterium mediated gene transfer. Observations based on GFP expression showed that miRNA159 targets and decreases the expression of GAMyb in vivo. v To conclude, this study can be evaluated as a distinctive study for two aspects / (i) it is the first study assessing a &ldquo / putative&rdquo / barley miRNA function biologically and (ii) developed a practical and effective functional assay for miRNA studies in plants.