Ethylene, a plant hormone, plays an essential role in many aspects about plant development, growth, ripening, and senescence. In addition, it also regulates several responses when plants suffer stress from drought, flood, herbivore bites, wound, etc. ACC synthase and ACC oxidase belong to two multigene families. In this study, PCR (polymerase chain reaction) and RACE (rapid amplification of cDNA ends) methods were used to amplify the ACC oxidase sequences in Cattleya bicolor orchid flower. The results show that there exists differences in the 3¡¦-UTR (untranslated region) of orchid gene sequences. Compare the ACC oxidase sequences, including the cDNA ORF (open reading frame) sequences and the amino acid sequences, of several different species, the sequence similarity among the three Laeliinae orchids, namely C.bicolor, C. intermedia, and Laelia anceps, is the highest. The similarity of cDNA ORF sequences and amino acid sequences between orchids and the other plants, such as rice, apple and torenia, is comparatively lower. It was proposed that the protein located in cytoplasma (or in mitochondrial matrix space), agrees with the result from analysis of amino acid hydrophilicity prediction. The ultimate goal of this study is to postpone the flower senescence by the way of plant transfection. In the present findings, it only deals with the cloning and analysis of the ACC oxidase genes in C. bicolor.

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Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-05T13:20:52Z No. of bitstreams: 1 texto completo.pdf: 754082 bytes, checksum: 9fbc505e90247c8c5cba11e335573762 (MD5) / Made available in DSpace on 2017-04-05T13:20:52Z (GMT). No. of bitstreams: 1 texto completo.pdf: 754082 bytes, checksum: 9fbc505e90247c8c5cba11e335573762 (MD5) Previous issue date: 2002-03-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / As mutações espontâneas na espécie Lycopersicon esculentum têm sido muito utilizadas pelos melhoristas como fonte de variabilidade genética com o objetivo de produzir tomates que apresentem maior resistência ao manuseio pós-colheita. Na região produtora de hortaliças de Viçosa, MG, identificaram-se plantas de tomate ‘Santa Clara’ cujos frutos apresentam coloração “amarelo-creme” quando imaturos e diversos aspectos de seu amadurecimento alterados. O objetivo do presente trabalho foi de estudar as alterações fisiológicas, visuais e ultraestruturais em frutos de tomateiro do cv. Santa Clara e seu mutante natural ‘Firme’ durante o amadurecimento na planta e o efeito da aplicação de etileno em frutos colhidos no estádio verde-maduro. Durante o amadurecimento na planta, os frutos ‘Santa Clara’ apresentaram mudança de cor da epiderme mais gradual e menos intensa do que os frutos mutantes, que apresentaram maior intensidade de cor vermelha nos últimos estádios de maturidade; observou-se aumento nos teores de carotenóides totais e licopeno de mais de 20 vezes tanto para frutos normais como para frutos mutantes. Os estudos de ultraestrutura evidenciaram que durante o amadurecimento de tomates ‘Santa Clara’ os cloroplastos pré-existentes se diferenciam em cromoplastos; por outro lado, no mutante ‘Firme’ a diferenciação em cromoplastos pode ocorrer inteiramente independente da presença de cloroplastos. Os frutos mutantes, durante o amadurecimento na planta, apresentaram menor produção de CO 2 e etileno em todos os estádios de amadurecimento; apesar da atividade da oxidase do ACC ter apresentado padrão de comportamento distinto durante o amadurecimento na planta entre frutos mutantes e normais, a magnitude da atividade foi a mesma. Frutos mutantes apresentaram atraso no aumento da atividade da enzima poligalacturonase em relação aos frutos normais. Os frutos normais acumularam açúcares durante seu amadurecimento na planta, enquanto que os frutos mutantes perderam açúcares com o amadurecimento, sendo que estes apresentaram teores de açúcares totais menores do que os de tomates normais tanto no pericarpo quanto no tecido locular. As mudanças fisiológicas características do amadurecimento dos frutos de tomate, como a perda de firmeza e a mudança de cor, foram influenciadas de maneira semelhante pela aplicação de etileno em frutos colhidos no estádio verde-maduro e armazenados a temperatura ambiente tanto para frutos normais como para frutos mutantes, o que indica que a sensibilidade do tecido ao etileno não foi alterada pela mutação. Os frutos mutantes apresentaram atraso na produção autocatalítica de etileno em relação aos frutos normais após a aplicação de etileno. A menor dose de etileno, 100 μL.L -1 , foi suficiente para acelerar o amadurecimento dos frutos do cv. Santa Clara assim como de seu mutante natural ‘Firme’. / Breeders have been using natural mutants of Lycopersicon esculentum species as source of genetic variability to enhance tomato fruit postharvest life. ‘Santa Clara’ tomato plants showing pale-yellow fruits and others fruit ripening aspects changed were found in Viçosa, MG. The aim of this work was to study the physiological, visual, and ultrastructural changes during ripening of ‘Santa Clara’ and ‘Firme’ fruits attached to the plant and the effect of exogenous ethylene on ripening of mature green fruit. ‘Santa Clara’ fruit showed a more gradual and less intense change on skin color than mutant fruit; we reported a rise higher than twenty fold in total carotenoids and lycopene levels during fruit ripening for ‘Santa Clara’ and ‘Firme’ fruits. Ultrastucture studies provide evidence that during ‘Santa Clara’ tomato fruit ripening chromoplast indeed differentiate from preexisting chloroplast; on the other hand, chromoplast differentiation in mutant fruit indicates that chromoplast development can be a process entirely independent of the chloroplast. Mutant fruit showed lower ethylene and CO 2 production at all maturity stages. ACC oxidase activity showed a distinct pattern during ripening of attached wild type and mutant fruits, nevertheless, the amount found for mutant and wild type were practically the same. Mutant fruit showed a delay on poligalacturonase activity rise comparing to wild type fruit. While wild type fruit showed a rise on total soluble sugars content during ripening mutant fruit showed a decrease on it, nevertheless, mutant fruit showed lower levels than wild type fruit for locular and pericarp total soluble sugars content at all maturity stages. Physiological changes during tomato fruit ripening, such as firmness loss and color change, were effected in a similar way by application of exogenous ethylene on mature green fruit stored at room temperature for mutant and wild type fruit, indicating that mutation did not change tissue ethylene-sensitivity. Mutant fruit showed a delay on autocatalytic ethylene production after application of exogenous ethylene when compared to ‘Santa Clara’ fruit. The lower ethylene concentration studied, 100 μLL -1 , enhanced ripening of ‘Santa Clara’ and ‘Firme’ fruits. / Tese importada do Alexandria

According to our previous results, ethephon-induced sweet potato leaf senescence and senescence-associated gene SPCP1 expression was affected by reduced glutathione, EGTA, and cycloheximide (Chen et al., 2009). These data suggest that calcium influx, reactive oxygen species (ROS) and de novo synthesized proteins can affect ethephon-mediated effects. Therefore, PCR-selective substractive hybridization and RACE-PCR methods were used to clone 5 full-length cDNAs encoded putative calmodulin (SPCAM), catalase (SPCATA), anionic peroxidase (SPPA), ACC oxidase (SPACO), and DSS1-like protein (SPDSS1) from mixed samples of ethephon-treated leaves for 6 and 24 hours. The ORF of SPCAM contains 450 nucleotides and encodes 149 amino acids. There are 4 putative EF-motifs in the deduced protein structure. SPCAM exhibited amino acid sequence identity with isolated Arabidopsis calmodulins from 48% to 100%, and was completely the same as CaM7 calmodulin. The ORF of SPCATA contains 1479 nucleotides and encodes 492 amino acids. SPCAM exhibited high amino acid sequence identity with other plant catalases from 71.2% to 80.9%, and had the highest identity with mangrove catalase. The ORF of SPPA contains 1068 nucleotides and encodes 355 amino acids. SPPA exhibited amino acid sequence identity with other published sweet potato peroxidase isoforms from 28.7% to 97.5%, and had the highest identity with anionic peroxidase SWPA4. The ORF of SPACO contains 930 nucleotides and encodes 309 amino acids. SPACO exhibited high amino acid sequence identity with other plant ACC oxidases from 62.3% to 81.5%, and had the highest identity with tobacco ACC oxidase. The ORF of SPDSS1 contains 228 nucleotides and encodes 75 amino acids. SPDSS1 exhibited amino acid sequence identity with other DSS1 from 25.2% to 62.3%, and had the highest identity with maize DSS1. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in natural senescent leaves. DAB staining showed that H2O2 amount was remarkably elevated at S3 senescent leaves compared to leaves of the other developmental stages. Evan blue staining also demonstrated that S3 senescent leaf had more cell death compared to S0 young leaves. In addition ethephon-induced leaf senescence exhibited similar results. The chlorophyll contents and Fv/Fm values were significantly reduced, however, the isolated gene expression was remarkably enhanced in ethephon-treated leaves compared to dark control. DAB staining showed that H2O2 amount was remarkably elevated at 72 hours in ethephon-treated leaves compared to dark control. Evan blue staining also demonstrated that ethephon-treated leaf for 72 hours had more cell death compared to dark control. Based on these data we conclude that SPCAM, SPCATA, SPPA, SPACO and SPDSS1 gene expression were significantly increased in natural and ethephon-induced senescent leaves. The possible functions of these isolated genes in association with events in ethephon-induced leaf senescence, including calcium influx, ROS elevation or scavenge, and following signaling will be discussed.

Ethylene (C2H4) is a very simple molecule, a gas, and has numerous effects on the growth, development and storage life of many fruits, vegetables and ornamental crops. In higher plants, ethylene is produced from L-methionine in essentially all tissues and ACC Synthase and ACC Oxidase are the two key enzymes in the biosynthesis of ethylene. The objective of the present study was to transform tobacco (Nicotiana tabacum L. cv. Samsun) plant with partial sequence of torenia acc oxidase gene in antisense and sense orientations via Agrobacterium-mediated gene transfer system, and to analyze its effect on ethylene production in transgenic plants. Six antisense and seven sense T0 putative transgenic lines were obtained and were further analyzed with several assays. Leaf disc assay and chlorophenol red assay under selection (75 mg/L kanamycin) revealed positive results compared to the non-transformed plant. T1 generations were obtained from all putative transgenic lines. PCR analysis and Northern Blot Hybridization results confirmed the transgenic nature of T1 progeny. Furthermore, ethylene amount produced by flowers were measured with gas chromatography, which resulted in an average of 77% reduction in S7 line and 72% reduction in A1 line compared with the control flowers. These results indicated that, transgenic tobacco plants carrying torenia acc oxidase transgene both in antisense and sense orientations showed reduced ethylene production thus a possibility of flower life extension.

Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_daiane_benemann.pdf: 778748 bytes, checksum: 5f3a1eba7b00cb2cf702e1d46bc5e43b (MD5) Previous issue date: 2008-08-18 / The objective of this work is to seek a protocol for regeneration of the melon (Cucumis melo L.) cv. Gaucho and subsequent genetic transformation of this fruit. To this end were conducted three experiments. In the first, the cotyledons were divided into 2, 3, 4 and 5 equal parts, and inoculated with MS medium containing 30 g L sucrose, 0.9 mg L-1 BAP, 0.3 mg L-1 ABA and 2.2 g L-1 CaCl2. It was observed that the number of cuts not influenced the percentage of explants regenerated. In the second experiment, using the same means of cultivation of the previous experiment, the cotyledons were submitted to different densities of flow of photons. It was found that the regenerative process was not affected by the absence or presence of light (2 and 21 μmol m-2 s-1), but when the explants remained in the dark had greater formation of callus. In the third experiment sought to establish conditions for seed germination and its effect on the regeneration under different concentrations of BAP. It was the highest rate of explants regenerated lower when the period of germination in the middle and lower the concentration of BAP in the regenerative. After obtaining the protocol for regeneration, was conducted test of sensitivity of explants the selection of antibiotic, setting up the concentration of 75 mg L-1, kanamycin as ideal to select the cells transformed. For the regeneration of the shoots explants were cultivated in MS medium containing 0.9 mg L-1 BAB, 0.3 mg L-1 ABA, 2.2 g L-1 CaCl2, 75 mg L-1 kanamycin and 250 mg L-1 cefotaxime. For the genetic transformation was used Agrobacterium tumefaciens LBA 4404, with a clone of ACC oxidase in antisense orientation, called pAP4as. However, it was not confirmed by inserting the sequence, the PCR test, the tissue analyzed. It was found that these studies were not described with Agrobacterium efficient to obtain seedlings containing the antisense gene of ACC oxidase. It was found that these studies were not described with Agrobacterium efficient to obtain seedlings containing the antisense gene of ACC oxidase. / O presente trabalho teve como objetivo desenvolver um protocolo eficiente de regeneração para o melão (Cucumis melo L.), cv. Gaúcho, visando posterior transformação genética do mesmo. Para tal fim foram realizados três experimentos. No primeiro, os cotilédones foram divididos em 2, 3, 4 e 5 partes iguais e, inoculados em meio MS contendo 30 g L-1 de sacarose, 0,9 mg L-1 BAP, 0,3 mg L-1 ABA e 2,2 g L-1 CaCl2. No segundo experimento, utilizando o mesmo meio de cultura do experimento anterior, os cotilédones foram submetidos a diferentes densidades de fluxo de fótons. No terceiro procurou-se estabelecer as condições para germinação das sementes e sua influência na regeneração cotiledonar sob diferentes concentrações de BAP. Observou-se que cotilédones seccionados em 5 partes apresentaram maior média de explantes regenerantes. Já os cotilédones que permaneceram sob intensidade luminosa de 2 μmol m-2 s-1 apresentaram maior média de explantes regenerantes e os que ficaram no escuro apresentaram maior média de explantes com calos e raiz. Observou-se também que quanto maior o período germinativo e maiores concentrações de BAP no meio regenerativo, menor é a taxa de explantes regenerantes. Após a obtenção do protocolo de regeneração, foi realizado teste de sensibilidade dos explantes ao antibiótico de seleção, determinando-se a concentração de 75 mg L-1 de canamicina como ideal para selecionar as células transformadas. Para a regeneração de brotações os explantes foram cultivados em meio MS contendo 0,9 mg L-1 BAP, 0,3 mg L-1 ABA, 2,2 g L-1 CaCl2, 75 mg L-1 canamicina e 250 mg L-1 cefotaxima. Para a transformação genética, foi utilizado Agrobacterium tumefaciens LBA 4404, com um clone da ACC oxidase em orientação antisense, denominado pAP4as. Entretanto, não foi confirmada a inserção da seqüência, pelo teste de PCR, nos tecidos analisados. Verificou-se que estes estudos descritos com Agrobacterium não foram eficientes para a obtenção de plântulas contendo o gene antisense da ACC oxidase.

L’ACC Oxydase est une enzyme à Fe(II) non-hémique impliquée dans la biosynthèse de l’éthylène chez les plantes. Notre compréhension du mécanisme ainsi le rôle des différents cofacteurs nécessite l’obtention des données structurales. Une structure cristallographique a été publiée montrant la partie C-terminale (C-term) éloignée du site actif. Ce n’est pas la conformation active car la partie C-term est essentielle à l’activité. Un modèle structural a été construit dans lequel la partie C-term est tournée vers le site actif. Différentes conformations semblent donc possibles. Le marquage de spin couplé à la spectroscopie RPE est une technique puissante pour sonder la dynamique structurale des protéines. Elle implique la liaison de nitroxydes sur des cystéines. Il est possible d’analyser la mobilité des sondes pour obtenir des informations sur leur environnement local. Par l’utilisation de techniques de RPE avancées, des mesures de distances entre deux sondes sont possibles. Des mutants portant une ou deux cystéines ont été conçus. La dynamique des mutants marqués a été étudiée in vitro par RPE. Par RPE impulsionnelle, des distances ont été mesurées pour l’ACCO en présence de différentes combinaisons de cofacteurs. Les distances expérimentales ont été comparées à celles prédites à partir des structures cristallographiques et du modèle structural et aussi à celles obtenues par des calculs de dynamique moléculaire. Pour cibler d’autres positions sur l’ACCO, l’introduction d’un acide aminé non naturel a été réalisée avec succès permettant d’obtenir de premières données structurales. Des données structurales préliminaires par RPE in cell sont également présentées / ACC Oxidase is a nonheme iron(II) containing enzyme involved in the biosynthesis of ethylene in plants. ACCO reaction mechanism and the role of the various cofactors are not well understood and structural and dynamic data are still required. A crystallographic structure has been reported showing the C-terminal part (C-term) away from the active site. This is not the active conformation as it has been shown that the C-term is essential. Later, a structural model has been proposed in which the C-term is folded towards the active site. Different conformations can be hypothesized. A technique well suited to monitor protein dynamics is site-directed spin labeling followed by EPR spectroscopy. It relies on the insertion of a nitroxide derivative on cysteines. Using this approach, it is possible to analyze the mobility of the label in order to obtain information on its local environment. Moreover using advanced EPR techniques, it is possible to acquire interspin distances between two incorporated probes. Mutants bearing one or two cysteines at desirable positions were designed. The dynamics of labeled mutants were studied in vitro using continuous wave EPR. By pulsed EPR, distances were recorded for ACCO in presence of different combinations of cofactors. The experimental distances were compared to the predicted ones obtained from the crystallographic and model structures, and also to the calculated ones obtained by molecular dynamic simulations. A successful introduction of an unnatural amino acid onto the sequence of ACCO was performed, allowing to obtain earliest results. The achievement of preliminary structural data by in cell EPR are also presented