Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2009. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2008. / Härtill 3 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2010. / Härtill 3 uppsatser.

O relógio biológico de plantas é uma rede regulatória de grande relevância para a adaptação dos organismos ao ambiente. Essa rede é composta por diversas vias de controle transcricional e pós-transcricional que se retroalimentam e geram ritmos biológicos. O controle exercido pelo relógio pode ser observado nos mais diversos aspectos da fisiologia e desenvolvimento de plantas. No presente projeto de pesquisa foi investigada a relação entre o relógio biológico e a sinalização por auxinas, uma classe de fitohormônios, em cana-de-açúcar. Foram utilizadas técnicas de expressão gênica, como RT-qPCR, para definição de um protocolo robusto de avaliação de respostas transcricionais a auxinas em plântulas de cana-de-açúcar geradas por organogênese direta. Após 1h da aplicação de 80 µM auxina sintética ácido 1-naftalenoacético, foi possível observar controle transcricional evidente exercido pela aplicação de auxina sobre alguns genes. Também foi observado variação na resposta obtida, dependendo do horário do ciclo circadiano em que o estímulo era oferecido. Esse fenômeno de controle temporal sobre a resposta a um estímulo é chamado gating, sendo de grande relevância para a atuação do relógio biológico de plantas. A partir dessas observações foram realizadas análises de expressão gênica em larga escala, usando oligoarranjos, para compreensão mais aprofundada da conexão entre o relógio biológico e a sinalização por auxinas em cana-de-açúcar. Entre os genes diferencialmente expressos após estímulo com auxina, foi verificado grande presença de genes relacionados a respostas contraestresse biótico. Além disso, as respostas observadas devem estar sobre o controle do relógio biológico de cana-de-açúcar. Diversos genes relacionados a combate a infecções, como quitinases e taumatinas, tiveram sua expressão alterada após aplicação de auxinas, sendo possível observar diferenças no padrão de expressão dos genes dependendo do horário em que auxina era aplicada. Dessa forma, o relógio biológico de cana-de-açúcar, a partir da sinalização por auxinas, deve exercer controle sobre as respostas a estresses bióticos nesse organismo. Os dados obtidos são inéditos e podem contribuir para o aumento da produtividade de cana-de-açúcar assim como para o desenvolvimento de novas ferramentas biotecnológicas focadas nesse cultivar, o qual apresenta grande relevância econômica / The circadian clock is a regulatory network with great relevance to fitness of plants. This network creates biological rhythms, influencing plants metabolism and their interaction with the environment. The clock is composed of interlocking feedback transcriptional and post-transcriptional pathways. In the presente study, we investigated the interconnection between circadian clock and signaling through auxins, a group of phytohormones with great impact to plant biology. Using RT-qPCR, it was established a protocol to measure transcriptional responses after synthetic auxin 1-naphtalenacetic acid (NAA) treatment. The biological material used was leaves of sugarcane plantlets generated by direct organogenesis. After 1h treatment with 80 µM NAA, we observed obvious transcriptional responses in sugarcane plantlets. It was also possible to detect alterations of transcriptional responses according to the moment when the stimulus was offered. This temporal control is called gating and is of great relevance to plant circadian clocks. We then performed transcriptomic analysis, using oligoarrays, to get a deeper understanding of the results obtained. Indeed, it was verified that auxin stimulus is connected to biotic stress transcriptional responses and that these responses are clock-controlled. Transcripts coding for proteins like chitinases and thaumatins, which are related to biotic stress responses, were differentially expressed after auxin treatment. Also, the response of most genes was daytime-dependent. We conclude that sugarcane circadian clock, through auxin signaling, might exert control under biotic stressresponses in sugarcane. The data obtained are novelty and may contribute to increase sugarcane productivity and/or to development of new biotechnological tools dedicated to this cultivar.

Light and phytohormones, such as cytokinins, have been known to play a pivotal role in numerous physiological processes in plant cells. Previous work in our laboratory has revealed the light- and cytokinin- modulated changes both in the levels of specific tRNA species and their modified nucleotide contents, in addition to the characterization of specific tRNAs and tRNA genes from higher plants. The plant hormone cytokinin, which is of particular interest to us has been implicated to be involved in processes such as induction of cell division, plastid biogenesis and delay of senescence. Ongoing work in our laboratory also points towards the role of Ca2+ as a second messenger in cytokinin mediated gene expression. With the objective of isolation of specific tRNA genes which could then be used as probes to study the light- and phytohormone- induced changes in the levels of respective functional mature tRNAs, a previously isolated clone containing a 6.6kb insert that hybridized with 3 end labeled cucumber total cellular tRNA was sequenced by the dideoxy chain termination method. Sequence analysis of the 6.6 kb DNA fragment has revealed a chloroplast genome DNA fragment containing the trnNGUU and trnRACG genes in addition to the genes coding for the ribosomal RNAs 4.5S, 5S and 23S as well as the protein coding genes ccsA (cytochrome c-synthesis) and ndhD(NADH plastoquinone oxidoreductase).These genes were found to be arranged in the order-23S-4.5S-5S-trnRACG-trnNGUU-ccsA-ndhD. This shows a divergence from the gene organization in the completely sequenced chloroplast genomes of other higher plant species such as tobacco, maize, rice and Arabidopsis, especially with regard to the absence of a highly conserved trnLUAG gene that has been shown to be present in the trnNGUU-ndhD intergenic region. The cucumber chloroplast trnNGUU and trnRACG genes have shown very high homology (>90%) whereas ccsA and ndhD show 50-61% similarity to corresponding genes from chloroplast genomes of other plant species. The relative levels of tRNAArg and tRNAAsn were determined by Northern analysis using the tRNA gene probes, in etiolated excised cucumber cotyledons treated with light or phytohormones, such as cytokinin (BA) and auxin (2,4-D). Light and phytohormones were found to significantly increase the levels of tRNAArg unlike in the case tRNAAsn where no significant changes in the levels were observed. This result points towards the regulation of relative levels of specific tRNA species by light and cytokinin so as to match the codon usage of the mRNA population during light- and cytokinin- induced plant development in cucumber. Northern analyses were also performed to monitor the relative transcript levels of the plastid encoded ccsA and ndhD in etiolated excised cucumber cotyledons treated with light or phytohormones. ccsA transcript levels were found to be significantly reduced in auxin treated cucumber cotyledons where as exogenous application of cytokinin to either dark-grown or light exposed cotyledons did not seem to have any pronounced effect. ndhD transcripts were found to be up-regulated by cytokinin treatment or light exposure in comparison to un-treated controls probably indicating a point of overlap in the light/ cytokinin mediated signal transduction pathways. Auxin treatment on the other hand was found to down-regulate ndhD transcript levels also. Recent studies from our laboratory have demonstrated the involvement of a calcium-dependent protein kinase(CDPK) in the cytokinin-signal transduction pathway associated with the induction of pathogenesis-related proteins (chitinase and β 1-3 modulation of nuclear-encoded CDPK transcripts in response to light and exogenously added phytohormones such as cytokinins and auxin. Towards this end, partial CDPK cDNAs were generated from Cucumis Sativus by RT-PCR using degenerate primers designed based on the conserved regions of the known CDPK proteins available in the database, cloned in pGEM-T and sequenced. Sequence analysis of twenty partial cDNA clones revealed the presence of at least four CDPK isoforms in Cucumis sativus (CuCDPK 1-4). Of the four partial CDPK cDNAs, the tissue-specific expression level of CuCDPK3 was studied using the highly sensitive Taqman Analysis (Quantitative RT-PCR). The results obtained indicate that, in excised dark-grown cucumber cotyledons light and cytokinin were found to up-regulate the levels of CuCDPK3 unlike auxin, which was found to have no significant effect. In cucumber hypocotyls, which had the highest levels of CuCDPK3, light was found to have a down-regulatory effect whereas cytokinin and auxin did not bring about any significant changes in the levels of CuCDPK3. In cucumber root tissue, both light and cytokinin were found to have a down-regulatory effect on the levels of CuCDPK3, unlike auxin. The southern analysis of cucumber genomic DNA revealed a CDPK multi-gene family in cucumber. Since cytokinins have been known to play a role in both etioplast and chloroplast biogenesis and since various groups have recently reported the presence of higher plant homologues of bacterial cell-division protein FtsZ and the requirement of plant nuclear-encoded FtsZs for plastid division, efforts were also made to isolate and to study the expression of cucumber FtsZ in dark-grown cucumber cotyledon tissue treated exogenously with light/phytohormones. Towards this end, a partial FtsZ cDNA was generated from cucumber by RT-PCR using degenerate primers designed based on conserved regions of known plant FtsZ proteins. Results of the Taqman Analysis indicate that cytokinin, unlike auxin, mimics the action of light by increasing the levels of CuFtsZ transcripts in dark-grown cotyledon tissue suggesting the involvement of FtsZ in cytokinin-induced plastid-biogenesis.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2008. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Peltophorum dubium (Sprengel) Taubert is a native forest species with many possible uses, with great potential for commercial plantations. However, there is little information on the production of seedlings of this species, which, together with the inherent limitations of tree species, impedes the setting of breeding programs and, therefore, the establishment of stands of appropriate genetic quality. Studies related to the Peltophorum dubium propagation by tissue culture techniques can help in this regard. Therefore, this study aimed to evaluate methodologies for in vitro culture of Peltophorum dubium, with the specific purpose of increasing rates of multiplication of the species and reduce leaf senescence. Furthermore, we tried to establish an efficient methodology for the indirect organogenesis, which can be an alternative way to induce organs. On in vitro multiplication and leaf senescence were investigated different sources and concentrations of cytokinins 6-benzylaminopurine (BAP), kinetin (KIN), isopentenyladenine (2iP), and thidiazuron (TDZ), combined one with each other, or isolated, and its association with α-naphthaleneacetic acid (NAA) and in the case of kinetin, 2iP and TDZ, also with BAP. In indirect organogenesis, we evaluated the effect of different concentrations of 2.4-D and various explants cultured in the presence or absence of light. It can be concluded that the use of cytokinin 2iP, in the evaluated concentrations in the presence of NAA or in association with NAA and BAP, has no effect on in vitro multiplication of the epicotyls containing cotyledonary node of Peltophorum dubium. Further, after 21 days of in vitro culture, under conditions of high temperature, occurs expressive leaf senescence. Cytokinins BAP, KIN, 2iP and TDZ, in evaluated concentrations, do not influence the in vitro multiplication of Peltophorum dubium. To maintain controlled leaf senescence when TDZ is used is dispensable using NAA, however, when the cytokinin 2iP is employed, the presence of NAA contributes to a reduction in the number of senescent leaves. Generally, from 21 days of in vitro culture the auxin and cytokinins used (NAA, 2iP and TDZ) start to have a significant effect on the occurrence of leaf senescence, which will depend on the combination of growth regulators. Can be also checked that the presence of TDZ, 2iP and NAA at the concentrations tested have no influence on survival, establishment and number of leaves formed in Peltophorum dubium during the initial in vitro culture of epicotyls containing cotyledonary node, have been observed high averages for these variables. Furthermore, the combination of NAA and TDZ has significant deleterious effect on in vitro survival of Peltophorum dubium explants during the first 30 days of subculture. This association also has significant deleterious effect on in vitro establishment and number of new shoots in shoot apical segments during first 30 days of subculture of Peltophorum dubium. The presence of 2iP and NAA at the concentrations tested doesn't have influence on survival, establishment, number of shoots and number of leaves formed in Peltophorum dubium during the first subculture of 30 days. The callogenesis is maximized in the presence of 20 μM of 2.4-D in cotyledon and hypocotyl segments. In the absence of 2.4-D hypocotyl are more efficient in callus formation. There is a high phenolic oxidation, but that, in general, does not impair the formation of callus, mainly in hypocotyls and cotyledon segments. There are 100% of root formation in cotyledon segments in the presence of light and nutritive medium supplemented with 40 μM of 2.4-D; in the absence of light and in the presence of 20 μM auxin rooting is also satisfactory. / Peltophorum dubium (Sprengel) Taubert é uma espécie florestal nativa com muitas possibilidades de uso e com grande potencial para plantios comerciais. No entanto, há poucas informações sobre a produção de mudas desta espécie, o que, aliado às limitações inerentes às espécies arbóreas, dificulta a implantação de programas de melhoramento genético e, consequentemente, o estabelecimento de povoamentos de adequada qualidade genética. Estudos relacionados à propagação de Peltophorum dubium por técnicas da cultura de tecidos in vitro podem auxiliar nesse sentido. Considerado o exposto, o presente estudo objetivou avaliar o efeito de fontes, concentrações e combinações de fitorreguladores na multiplicação e senescência foliar in vitro de Peltophorum dubium. Adicionalmente, procurou-se estudar a resposta de diferentes explantes em relação à organogênese indireta, que pode ser uma maneira alternativa para a indução de órgãos. Na multiplicação in vitro e na senescência foliar in vitro foram avaliadas diferentes fontes e concentrações das citocininas 6-benzilaminopurina (BAP), cinetina (CIN), isopenteniladenina (2iP) e thidiazuron (TDZ), combinadas entre si, ou isoladas, bem como sua associação com ácido α-naftalenoacético (ANA) e, no caso de CIN, 2iP e TDZ, com BAP. Na organogênese indireta, avaliou-se o efeito de diferentes concentrações de 2,4-D e diferentes explantes cultivados na presença ou ausência de luz. Pode-se concluir que o emprego da citocinina 2iP, nas concentrações testadas, na presença de ANA ou em conjunto com ANA e BAP, não exerce influência na multiplicação in vitro de epicótilos contendo o nó cotilenodar de Peltophorum dubium. Ainda, aos 21 dias de cultivo in vitro, em condições de temperatura elevada, ocorre expressiva senescência foliar. As citocininas BAP, CIN, TDZ e 2iP, nas concentrações testadas, não influenciam na multiplicação in vitro de Peltophorum dubium. Verificou-se que, quando se utiliza TDZ, é dispensável a utilização de ANA para que se mantenha controlada a senescência foliar, no entanto, quando é 2iP, a presença de ANA contribui para a redução no número de folhas senescentes. De maneira geral, a partir dos 21 dias de cultivo in vitro, a auxina e as citocininas utilizadas (ANA, 2iP e TDZ) passam a exercer efeito significativo na ocorrência de senescência foliar, a qual vai depender da combinação dos fitorreguladores. Pode-se verificar, também, que a presença de TDZ, ANA e 2iP, nas concentrações testadas, não exerce influência na sobrevivência, estabelecimento e número de folhas formadas em Peltophorum dubium durante o cultivo in vitro inicial de epicótilos contendo o nó cotiledonar, observando-se elevadas médias para estas variáveis. Ainda, a associação entre ANA e TDZ tem efeito prejudicial significativo na sobrevivência in vitro de explantes de Peltophorum dubium durante o primeiro subcultivo de 30 dias. Esta associação também tem efeito prejudicial significativo no estabelecimento in vitro e número de brotos formados em segmentos apicais caulinares de Peltophorum dubium durante o primeiro subcultivo de 30 dias. A presença de 2iP e ANA, nas concentrações testadas, não exerce influência na sobrevivência, estabelecimento, número de brotos e número de folhas formados em Peltophorum dubium durante o primeiro subcultivo de 30 dias. A calogênese é maximizada na presença de 20 μM de 2,4-D em segmentos cotiledonares e hipocótilos. Na ausência de 2,4-D os hipocótilos são mais eficientes na calogênese. Observam-se altas porcentagens de oxidação fenólica, mas de maneira geral, as oxidações não inviabilizam a formação de calos, principalmente em segmentos cotiledonares e hipocótilos. Ocorrem 100% de formação de raízes em segmentos cotiledonares, na presença de luz e em meio nutritivo suplementado com 40 μM de 2,4-D; na ausência de luz e presença de 20 μM da auxina, a rizogênese é também satisfatória.

O relógio biológico de plantas é uma rede regulatória de grande relevância para a adaptação dos organismos ao ambiente. Essa rede é composta por diversas vias de controle transcricional e pós-transcricional que se retroalimentam e geram ritmos biológicos. O controle exercido pelo relógio pode ser observado nos mais diversos aspectos da fisiologia e desenvolvimento de plantas. No presente projeto de pesquisa foi investigada a relação entre o relógio biológico e a sinalização por auxinas, uma classe de fitohormônios, em cana-de-açúcar. Foram utilizadas técnicas de expressão gênica, como RT-qPCR, para definição de um protocolo robusto de avaliação de respostas transcricionais a auxinas em plântulas de cana-de-açúcar geradas por organogênese direta. Após 1h da aplicação de 80 µM auxina sintética ácido 1-naftalenoacético, foi possível observar controle transcricional evidente exercido pela aplicação de auxina sobre alguns genes. Também foi observado variação na resposta obtida, dependendo do horário do ciclo circadiano em que o estímulo era oferecido. Esse fenômeno de controle temporal sobre a resposta a um estímulo é chamado gating, sendo de grande relevância para a atuação do relógio biológico de plantas. A partir dessas observações foram realizadas análises de expressão gênica em larga escala, usando oligoarranjos, para compreensão mais aprofundada da conexão entre o relógio biológico e a sinalização por auxinas em cana-de-açúcar. Entre os genes diferencialmente expressos após estímulo com auxina, foi verificado grande presença de genes relacionados a respostas contraestresse biótico. Além disso, as respostas observadas devem estar sobre o controle do relógio biológico de cana-de-açúcar. Diversos genes relacionados a combate a infecções, como quitinases e taumatinas, tiveram sua expressão alterada após aplicação de auxinas, sendo possível observar diferenças no padrão de expressão dos genes dependendo do horário em que auxina era aplicada. Dessa forma, o relógio biológico de cana-de-açúcar, a partir da sinalização por auxinas, deve exercer controle sobre as respostas a estresses bióticos nesse organismo. Os dados obtidos são inéditos e podem contribuir para o aumento da produtividade de cana-de-açúcar assim como para o desenvolvimento de novas ferramentas biotecnológicas focadas nesse cultivar, o qual apresenta grande relevância econômica / The circadian clock is a regulatory network with great relevance to fitness of plants. This network creates biological rhythms, influencing plants metabolism and their interaction with the environment. The clock is composed of interlocking feedback transcriptional and post-transcriptional pathways. In the presente study, we investigated the interconnection between circadian clock and signaling through auxins, a group of phytohormones with great impact to plant biology. Using RT-qPCR, it was established a protocol to measure transcriptional responses after synthetic auxin 1-naphtalenacetic acid (NAA) treatment. The biological material used was leaves of sugarcane plantlets generated by direct organogenesis. After 1h treatment with 80 µM NAA, we observed obvious transcriptional responses in sugarcane plantlets. It was also possible to detect alterations of transcriptional responses according to the moment when the stimulus was offered. This temporal control is called gating and is of great relevance to plant circadian clocks. We then performed transcriptomic analysis, using oligoarrays, to get a deeper understanding of the results obtained. Indeed, it was verified that auxin stimulus is connected to biotic stress transcriptional responses and that these responses are clock-controlled. Transcripts coding for proteins like chitinases and thaumatins, which are related to biotic stress responses, were differentially expressed after auxin treatment. Also, the response of most genes was daytime-dependent. We conclude that sugarcane circadian clock, through auxin signaling, might exert control under biotic stressresponses in sugarcane. The data obtained are novelty and may contribute to increase sugarcane productivity and/or to development of new biotechnological tools dedicated to this cultivar.

With new 2,4-Dichlorophenoxyacetic acid (2,4-D) tolerant crops, increases in off-target movement events are expected. New formulations may mitigate these events, but standard lab techniques are ineffective in identifying these 2,4-D formulations. Using Fourier-transform infrared spectroscopy and machine learning algorithms, research was conducted to classify 2,4-D formulations in treated herbicide-tolerant soybeans and cotton and observe the influence of leaf treatment status and collection timing on classification accuracy. Pooled Classification models using k-nearest neighbor classified 2,4-D formulations with over 65% accuracy in cotton and soybean. Tissue collected 14 DAT and 21 DAT for cotton and soybean respectively produced higher accuracies than the pooled model. Tissue directly treated with 2,4-D also performed better than the pooled model. Lastly, models using timing and treatment status as factors resulted in higher accuracies, with cotton 14 DAT New Growth and Treated models and 28 DAT and 21 DAT Treated soybean models achieving the best accuracies.