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Apesar de sua elevada importância econômica mundial, o cultivo do tomateiro é classificado como de alto risco devido à infestação da cultura por um grande número de pragas e doenças. Esse problema leva tal cultura a depender amplamente da aplicação de agroquímicos que, além de elevaram o custo de produção, são potenciais causadores de danos ambientais e do aparecimento de outras pragas e doenças. Observando o problema mais detalhadamente, pode-se perceber que uma grande parcela das perdas advém dos danos causados por artrópodes herbívoros, os quais são capazes de se alimentar de diversas partes da planta, causando, geralmente, perda de produtividade. Apesar disso, o tomateiro apresenta mecanismos de defesa naturais contra tais pragas, como tricomas e aleloquímicos, que agem intoxicando os artrópodes, dificultando sua movimentação e/ou alterando varias fases de seu desenvolvimento. A busca por genótipos de tomateiro com maior densidade de tricomas e elevado teor de aleloquímicos é hoje considerado um hot spot de pesquisa, devido ao benefício que trariam como redução do custo de produção e maior produtividade. Sabese que vários hormônios vegetais estão ligados a geração de caracteres anti-herbivoria, mas, atualmente, um grande foco vem sendo dado somente ao ácido jasmônico, devido ao seu claro papel na formação de tricomas, aleloquímicos, inibidores de proteases, dentre outros. Visando mostrar a importância de uma abordagem multi-hormonal nesse tipo de estudo, o presente trabalho teve como objetivo avaliar quais hormônios vegetais estão envolvidos com a formação de vários caracteres anti-herbivoria em tomateiro, como densidade de tricomas e teor de aleloquímicos e inibidores de protease. Para tal, fez-se uso da um grande número de mutantes hormonais introgredidos na cultivar Micro-Tom (a qual apresenta diversas facilidades de trabalho como porte reduzido e rápido ciclo de vida). Demonstrou-se que, apesar do ácido jasmônico ser extremamente importante na formação de tais caracteres, outros hormônios também atuam fortemente em tal papel. Etileno, giberelinas e auxina alteram de forma indireta a densidade de tricomas em tomateiro, através de alteração na área de células epidérmicas. O ácido jasmônico é um forte regulador positivo da formação de tricomas, do aleloquímico zingibereno e da formação de inibidores de protease. De forma antagônica, brassinosteróides parecem controlar negativamente a densidade de tricomas, a produção de zingibereno e inibidores de protease. Interessantemente, observou-se que esse controle negativo efetuado por brassinosteróides acontece através de um controle na via do ácido jasmônico, o qual foi comprovado pela produção de duplos mutantes. Resultados obtidos em testes com o herbívoro polífago Spodoptera frugiperda e com a praga de tomate Tuta absoluta (traça-do-tomateiro), bem como de análise de expressão gênica comprovaram a importância do ácido jasmônico, brassinosteróides e sua interação na defesa a herbivoria. Os resultados aqui apresentados sugerem que o foco para futuros estudos da formação de mecanismos anti-herbivoria em tomateiro deve ser voltado não só para o ácido jasmônico como para brassinosteroides e para suas ações antagônicas. / Even though the cultivation of tomato has a high economical importance, this culture is classified as being of elevated risk because of it common infestation by pests and diseases. This problem makes this culture greatly dependent of the application of several agrochemicals, which leads to increase in cost of production, environmental damages and also facilitating the appearance of new pests and diseases. Looking carefully to the problem, it is possible to conclude that a great part of tomato losses are caused by herbivory-arthropods, which are capable of feeding of several parts of the plant, causing, generally, losses in the productivity. However, tomato has natural defense mechanisms against those pests, for example trichomes and allelochemicals, which act poisoning the arthropods, hindering their movement through the plant and/or altering steps of their development. The search for tomato genotypes with great density of trichomes and elevated levels of allelochemicals is nowadays being considered as a hot spot of research, because of the benefits it would provide (for example: a decrease in the cost of production and also a higher productivity). Although it is believed that many plant hormones are involved in the generation of anti-herbivory traits, a great focus has been given only to jasmonic acid because of it clear action in the formation of those traits, such as trichomes, allelochemicals, proteinase inhibitors and many others. The present work objective was to evaluate which hormones are involved in the formation of anti-herbivory traits such as trichome density and allelochemicals and proteinase inhibitors content. For this reason, we made use of several hormonal mutants already introgressed in the Micro-Tom cultivar (which presents benefits as small size and fast life cycle). It was showed that jasmonic acid is an important hormone in the formation of these traits, but other phytohormones also play important roles. Ethylene, gibberellins and auxin alters trichome density indirectly, by altering the area of epidermal cells and thus cell number. Jasmonic acid is a positive regulator of trichome formation, the allelochemicals zingiberene content and also in the formation of proteinase inhibitors. However, brassinosteroids acts by negatively controlling all of those. Interestingly, we observed that this negative control happens through the control of jasmonic acid pathway, a hormonal interaction that was proved by double-mutant analysis. Results obtained in tests with the polyphagous insect Spodoptera frugiperda and the tomato specific pest Tuta absoluta (tomato pinworm) and also with qPCR showed the importance of jasmonic acid, brassinosteroid and their interaction in herbivory defense. Our data suggest that future studies about the formation of antiherbivory traits should be analyzed by focusing the functions not only of jasmonic acid but also brassinosteroid and their antagonist functions.

Apesar de sua elevada importância econômica mundial, o cultivo do tomateiro é classificado como de alto risco devido à infestação da cultura por um grande número de pragas e doenças. Esse problema leva tal cultura a depender amplamente da aplicação de agroquímicos que, além de elevaram o custo de produção, são potenciais causadores de danos ambientais e do aparecimento de outras pragas e doenças. Observando o problema mais detalhadamente, pode-se perceber que uma grande parcela das perdas advém dos danos causados por artrópodes herbívoros, os quais são capazes de se alimentar de diversas partes da planta, causando, geralmente, perda de produtividade. Apesar disso, o tomateiro apresenta mecanismos de defesa naturais contra tais pragas, como tricomas e aleloquímicos, que agem intoxicando os artrópodes, dificultando sua movimentação e/ou alterando varias fases de seu desenvolvimento. A busca por genótipos de tomateiro com maior densidade de tricomas e elevado teor de aleloquímicos é hoje considerado um hot spot de pesquisa, devido ao benefício que trariam como redução do custo de produção e maior produtividade. Sabese que vários hormônios vegetais estão ligados a geração de caracteres anti-herbivoria, mas, atualmente, um grande foco vem sendo dado somente ao ácido jasmônico, devido ao seu claro papel na formação de tricomas, aleloquímicos, inibidores de proteases, dentre outros. Visando mostrar a importância de uma abordagem multi-hormonal nesse tipo de estudo, o presente trabalho teve como objetivo avaliar quais hormônios vegetais estão envolvidos com a formação de vários caracteres anti-herbivoria em tomateiro, como densidade de tricomas e teor de aleloquímicos e inibidores de protease. Para tal, fez-se uso da um grande número de mutantes hormonais introgredidos na cultivar Micro-Tom (a qual apresenta diversas facilidades de trabalho como porte reduzido e rápido ciclo de vida). Demonstrou-se que, apesar do ácido jasmônico ser extremamente importante na formação de tais caracteres, outros hormônios também atuam fortemente em tal papel. Etileno, giberelinas e auxina alteram de forma indireta a densidade de tricomas em tomateiro, através de alteração na área de células epidérmicas. O ácido jasmônico é um forte regulador positivo da formação de tricomas, do aleloquímico zingibereno e da formação de inibidores de protease. De forma antagônica, brassinosteróides parecem controlar negativamente a densidade de tricomas, a produção de zingibereno e inibidores de protease. Interessantemente, observou-se que esse controle negativo efetuado por brassinosteróides acontece através de um controle na via do ácido jasmônico, o qual foi comprovado pela produção de duplos mutantes. Resultados obtidos em testes com o herbívoro polífago Spodoptera frugiperda e com a praga de tomate Tuta absoluta (traça-do-tomateiro), bem como de análise de expressão gênica comprovaram a importância do ácido jasmônico, brassinosteróides e sua interação na defesa a herbivoria. Os resultados aqui apresentados sugerem que o foco para futuros estudos da formação de mecanismos anti-herbivoria em tomateiro deve ser voltado não só para o ácido jasmônico como para brassinosteroides e para suas ações antagônicas. / Even though the cultivation of tomato has a high economical importance, this culture is classified as being of elevated risk because of it common infestation by pests and diseases. This problem makes this culture greatly dependent of the application of several agrochemicals, which leads to increase in cost of production, environmental damages and also facilitating the appearance of new pests and diseases. Looking carefully to the problem, it is possible to conclude that a great part of tomato losses are caused by herbivory-arthropods, which are capable of feeding of several parts of the plant, causing, generally, losses in the productivity. However, tomato has natural defense mechanisms against those pests, for example trichomes and allelochemicals, which act poisoning the arthropods, hindering their movement through the plant and/or altering steps of their development. The search for tomato genotypes with great density of trichomes and elevated levels of allelochemicals is nowadays being considered as a hot spot of research, because of the benefits it would provide (for example: a decrease in the cost of production and also a higher productivity). Although it is believed that many plant hormones are involved in the generation of anti-herbivory traits, a great focus has been given only to jasmonic acid because of it clear action in the formation of those traits, such as trichomes, allelochemicals, proteinase inhibitors and many others. The present work objective was to evaluate which hormones are involved in the formation of anti-herbivory traits such as trichome density and allelochemicals and proteinase inhibitors content. For this reason, we made use of several hormonal mutants already introgressed in the Micro-Tom cultivar (which presents benefits as small size and fast life cycle). It was showed that jasmonic acid is an important hormone in the formation of these traits, but other phytohormones also play important roles. Ethylene, gibberellins and auxin alters trichome density indirectly, by altering the area of epidermal cells and thus cell number. Jasmonic acid is a positive regulator of trichome formation, the allelochemicals zingiberene content and also in the formation of proteinase inhibitors. However, brassinosteroids acts by negatively controlling all of those. Interestingly, we observed that this negative control happens through the control of jasmonic acid pathway, a hormonal interaction that was proved by double-mutant analysis. Results obtained in tests with the polyphagous insect Spodoptera frugiperda and the tomato specific pest Tuta absoluta (tomato pinworm) and also with qPCR showed the importance of jasmonic acid, brassinosteroid and their interaction in herbivory defense. Our data suggest that future studies about the formation of antiherbivory traits should be analyzed by focusing the functions not only of jasmonic acid but also brassinosteroid and their antagonist functions.

published_or_final_version / abstract / Botany / Master / Master of Philosophy

by Chi-Ming Wong. / Bibliography: leaves 183-193 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

A multiplicação de orquídeas in vitro vem sendo utilizada há algum tempo com objetivo de elevar a taxa de multiplicação, além de eliminar patógenos e reduzir gastos na produção. Esta ferramenta de trabalho vem sendo rotineiramente utilizada no nosso laboratório, ao longo de mais de duas décadas em nosso laboratório, em pesquisas básicas de fisiologia e de aprimoramento da técnica de clonagem, principalmente de orquídeas. Neste caso,o uso da técnica visa a obtenção de maior estabilidade genética dos regenerantes em cultivos de longa duração. Plantas do gênero Catasetum apresentam atividade indeterminada do meristema apical caulinar quando incubadas no escuro, originando em pouco tempo longos estolões com crescimento indeterminado, comportamento raro no reino vegetal. Cada nó do caule estiolado possui uma gema lateral que, quando isolada e incubada no claro, forma rapidamente uma planta completa, facilitando a micropropagação. Outras espécies de orquídeas valorizadas na floricultura não apresentam tal facilidade na multiplicação, mostrando-se recalcitrantes à micropropagação, como é o caso do gênero Dendrobium (Orchidaceae). O objetivo deste estudo foi obter uma melhor compreensão dos mecanismos fisiológicos envolvidos no estiolamento de plantas Dendrobium \"Second Love\", que apresenta crescimento caulinar limitado quando sob ausência de luz, buscando compreender os efeitos do escuro e dos hormônios etileno e giberelina, bem como do radical livre óxido nítrico e gás carbônico na atividade dos meristemas apicais e laterais dessa orquídea. Como objetivo complementar, buscou-se estimular um estiolamento mais pronunciado, visando com isto um aumento potencial na formação de gemas laterais, paralelamente à quebra da dominância apical e o crescimento subsequente dos estolões. As plantas de Dendrobium utilizadas faziam parte do nosso estoque de germoplasma in vitro. Após 120 dias de incubação no claro, as plantas foram transferidas para o escuro e tratadas com diferentes concentrações de ácido giberélico (GA), paclobutrazol (PA - inibidor de biossíntese de giberelina), etileno, 1-metilciclopropeno (1-MCP - inibidor da ação do etileno) e óxido nítrico (NO). Análises mensais dos teores de etileno e CO2 acumulados nos frascos foram realizadas por meio de cromatografia gasosa durante três meses. Após 30, 60 e 90 dias de tratamento no escuro, quantificou-se o número de gemas laterais presentes nos estolões, o número de gemas laterais e apicais que se desenvolviam, o tamanho dos estolões formados, bem como os respectivos valores de massas fresca e seca destes. Por fim, buscou-se avaliar ainda a importância da incubação na penumbra e no escuro sobre o crescimento caulinar, o número de gemas laterais e o desenvolvimento destas após três meses de cultivo. O crescimento no escuro dos caules das plantas de Dendrobium \"Second Love\" mostrou-se extremamente lento e limitado quando comparado ao das plantas de Catasetum fimbriatum. No entanto, quando tratadas com 1.000 μM de óxido nítrico, verificou-se ao final do terceiro mês que o número de gemas laterais era cinco vezes maior do que nas respectivas plantas controle. O tratamento com 10 ppm de etileno apresentou um aumento significativo no número de gemas e de estolões laterais, quando comparados ao controle a partir do segundo mês de incubação. Quanto ao tamanho do estolão apical, os tratamentos com 5 e 50 μM de GA não apresentaram nenhum efeito promotor sobre alongamento caulinar. Mesmo não apresentando a retomada da atividade meristemática apical, o tratamento com 5 μM de PA liberou um número maior de estolões laterais que o controle. Plantas tratadas com 1.000 ppm de NO, a partir do segundo mês de incubação, apresentaram um número elevado de estolões laterais, além dos mesmos apresentarem-se significativamente maiores. O tratamento com 100 ppm de 1-MCP apresentou o mesmo fenótipo das plantas tratadas no claro, ou seja, não estiolaram mesmo sob a ausência de luz. Quanto à emissão de etileno, observou-se que o tratamento com 1-MCP acarretou um aumento significativo na emissão deste gás pela planta, alcançando valores vinte vezes maiores do que no tratamento controle. Já a emissão de CO2 foi menor no tratamento claro quando comparada a maioria dos outros tratamentos no escuro. Os tratamentos em maiores concentrações de GA e NO pareceram promover algum tipo de estresse na planta (evidenciado pela necrose dos tecidos), demonstrando que a espécie em questão pode ser sensível à níveis elevados destas substâncias / In vitro multiplication has been used for some time in order to improve multiplication rate, eliminate pathogens and reduce the production costs. This working tool has been routinely used in our laboratory for over than two decades of basic research in plant physiology and enhancement of cloning technique, especially orchids, aiming to obtain greater genetic stability of regenerants in long term crops. Genus Catasetum present indeterminate shoot apical meristem activity when incubated in the dark, resulting, in a short period of time, long stolons with indeterminate growth: rare behavior in the plant kingdom. Each etiolated steam node has a lateral bud that, when isolated and incubated in light, quickly forms a complete plant, facilitating micropropagation. Other species of valued orchids in floriculture, such as genus Dendrobium (Orchidaceae), have no such facility in multiplication, being recalcitrant to micropropagation. The goals of this study were to gain a better understanding of the physiological mechanisms involved in plant etiolation in Dendrobium \"Second Love\", which has limited stem growth when in dark: and to understand the effects of dark, gibberellin and ethylene (plant hormones), as well as the free radical nitric oxide and carbon dioxide in the activity of apical and lateral meristems of the orchid. As a complementary objective, we tried to stimulate etiolation, aiming to potentially increase lateral buds formation and to break apical dominance with a subsequent stolons growth. Dendrobium plants used in this work were part of our in vitro germplasm stock. After 120 days of incubation in light, the plants were transferred to dark and treated with different concentrations of gibberellic acid (GA), paclobutrazol (PA - gibberellin biosynthesis inhibitor), ethylene, 1-methylcyclopropene (1-MCP - ethylene action inhibitor) and nitric oxide (NO). During a three months period, monthly analyzes of the accumulated levels of ethylene and CO2 in the flasks were performed using gas chromatography. After 30, 60 and 90 days of dark treatment the number of lateral buds presented in stolons, the number of developed lateral and apical buds, the size of formed stolons, and the respective amounts of fresh and dry mass were quantified. Finally, we evaluated the importance of incubation to steam growth in low light and in the dark, and the number of lateral buds and their development after three months of incubation. The Dendrobium \"Second Love\" steam growth in dark is extremely slow and limited when compared to Catasetum fimbriatum plants. However, after three months of treatment with 1.000 μM nitric oxide it was found to have five times more lateral buds than the respective control treatment plants. The treatment with 10 ppm ethylene showed a significant increase in the number of buds and lateral stolons compared to the control treatment from the second month of incubation. Treatments with 5 and 50 μM of GA had no promoting effect on the apical stolon stem elongation. Although not presenting the resumption of apical meristem activity, 5 μM of PA treatment has released a greater number of lateral stolons than the control treatment. Plants treated with 1000 ppm of NO, from the second month of incubation, showed a higher number of lateral stolons, moreover they were significantly larger. Treatment with 100 ppm 1-MCP had the same phenotype as plants treated in light: in other words, they did not etiolate even in light absence. Regarding the ethylene emission, we observed that the treatment with 1-MCP caused a significant increase in the emission of this gas by the plant, reaching values twenty times higher than the control treatment. CO2 emission was lower in light treatment when compared to most of the other treatments in dark. Treatments at higher concentrations of NO and GA seemed to foster some sort of plant stress (evidenced by tissue necrosis), demonstrating that the specie in question may be sensitive to high levels of these substances

1v. (various paging) : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D.Sc.)--University of Adelaide, Dept. of Botany, 1978

<p>Plant natriuretic peptides (PNPs) are a novel class of extracellular, systemically mobile molecules that elicit a number of plant responses important in homeostasis and growth. Natriuretic peptides were first identified in vertebrates where they play a role in the regulation of salt and water balance. Subsequent experimental investigations have identified the presence of a natriuretic peptide hormone system in plants. While PNPs have been implicated in various physiological responses such as stomatal guard cell movements and regulation of net water uptake, its biological role has remained elusive. Here we have used co-expression and promoter content analysis tools to understand the biological role of the Arabidopsis thaliana PNP (AtPNP-A). The analysis of AtPNP-A and its co-expressed genes revealed that genes annotated as part of the systemic acquired resistance (SAR) pathway were over-represented, thus suggesting that AtPNP-A may function as a component of plant defense responses and specifically, SAR. The results further show that AtPNP-A shares many characteristics with pathogenesis related (PR) proteins in that its transcription is strongly induced in response to pathogen challenges, thus implying a newly described role for AtPNP-A in pathogen attack. Additional tissue expression analysis also indicated distinct localization of PNP activity in sepals and transcriptional meta-analysis showed that AtPNP-A may play a role in starch breakdown. Therefore, together with the finding that AtPNP-A plays a role in regulating phloem transport, we also hypothesize that AtPNP-A may play a role in phloem unloading in sepals to assist processes such as seed formation in plants. In plants, the second messenger, guanosine 3&rsquo / ,5&rsquo / -cyclic monophosphate (cGMP) mediates a whole range of important processes including salinity tolerance, disease resistance, drought tolerance and responses to light. Since PNPs regulate water and salt homeostasis via a cGMP-dependent signaling pathways, it is thus important to analyse the transcriptome induced by the second messenger (cGMP) in Arabidopsis thaliana to give a better understanding of its mechanism of action. This study was also supplemented by the analysis of the gibberellic acid (GA) dependent transcriptome, since cGMP also plays a role its transcription pathway. This data analysis, together with promoter content investigation, revealed that genes upregulated after cGMP treatment and down-regulated in the GA insensitive mutant (ga1-3) were enriched with a GA response element (GARE), while no GARE enrichment were observed in genes up-regulated in the ga1-3 mutant. These findings suggest that GARE is indicative of GA-induced and cGMP-dependent transcriptional up-regulation. Gene ontology analysis confirmed previous reports that cGMP is involved in ion homeostasis and indicated that the transcriptional cGMP response is bi-polar in the sense that both genes up- and down-regulated in response to cGMP is involved in cation transport. Additionally, ab initio analysis of genes transcriptionally dependent on cGMP identified CHX8 as a hub gene and promoter content of CHX8 co-expressed genes show enrichment of the GARE motif. The fact that CHX8 has its highest expression levels during male gametogenesis and pollen tube growth, together with our findings, suggest that GA-induced and cGMP- dependent genes may play a key role in ion and water homeostasis in the male gametophyte. Finally, we propose that the type of analysis undertaken here can yield new insights into gene regulation networks and inform experimental strategies to unravel complex transcription regulatory systems under different developmental and stimulus specific conditions.</p>

<p>Plant natriuretic peptides (PNPs) are a novel class of extracellular, systemically mobile molecules that elicit a number of plant responses important in homeostasis and growth. Natriuretic peptides were first identified in vertebrates where they play a role in the regulation of salt and water balance. Subsequent experimental investigations have identified the presence of a natriuretic peptide hormone system in plants. While PNPs have been implicated in various physiological responses such as stomatal guard cell movements and regulation of net water uptake, its biological role has remained elusive. Here we have used co-expression and promoter content analysis tools to understand the biological role of the Arabidopsis thaliana PNP (AtPNP-A). The analysis of AtPNP-A and its co-expressed genes revealed that genes annotated as part of the systemic acquired resistance (SAR) pathway were over-represented, thus suggesting that AtPNP-A may function as a component of plant defense responses and specifically, SAR. The results further show that AtPNP-A shares many characteristics with pathogenesis related (PR) proteins in that its transcription is strongly induced in response to pathogen challenges, thus implying a newly described role for AtPNP-A in pathogen attack. Additional tissue expression analysis also indicated distinct localization of PNP activity in sepals and transcriptional meta-analysis showed that AtPNP-A may play a role in starch breakdown. Therefore, together with the finding that AtPNP-A plays a role in regulating phloem transport, we also hypothesize that AtPNP-A may play a role in phloem unloading in sepals to assist processes such as seed formation in plants. In plants, the second messenger, guanosine 3&rsquo / ,5&rsquo / -cyclic monophosphate (cGMP) mediates a whole range of important processes including salinity tolerance, disease resistance, drought tolerance and responses to light. Since PNPs regulate water and salt homeostasis via a cGMP-dependent signaling pathways, it is thus important to analyse the transcriptome induced by the second messenger (cGMP) in Arabidopsis thaliana to give a better understanding of its mechanism of action. This study was also supplemented by the analysis of the gibberellic acid (GA) dependent transcriptome, since cGMP also plays a role its transcription pathway. This data analysis, together with promoter content investigation, revealed that genes upregulated after cGMP treatment and down-regulated in the GA insensitive mutant (ga1-3) were enriched with a GA response element (GARE), while no GARE enrichment were observed in genes up-regulated in the ga1-3 mutant. These findings suggest that GARE is indicative of GA-induced and cGMP-dependent transcriptional up-regulation. Gene ontology analysis confirmed previous reports that cGMP is involved in ion homeostasis and indicated that the transcriptional cGMP response is bi-polar in the sense that both genes up- and down-regulated in response to cGMP is involved in cation transport. Additionally, ab initio analysis of genes transcriptionally dependent on cGMP identified CHX8 as a hub gene and promoter content of CHX8 co-expressed genes show enrichment of the GARE motif. The fact that CHX8 has its highest expression levels during male gametogenesis and pollen tube growth, together with our findings, suggest that GA-induced and cGMP- dependent genes may play a key role in ion and water homeostasis in the male gametophyte. Finally, we propose that the type of analysis undertaken here can yield new insights into gene regulation networks and inform experimental strategies to unravel complex transcription regulatory systems under different developmental and stimulus specific conditions.</p>

Thesis (M.S.)--Oregon State University, 2008. / Printout. Accompanied by zipped folders that include Excel and pdf files. Includes bibliographical references. Also available on the World Wide Web.