ABA insensitive mutants (abi1 - abi5) have been isolated in germination screens that use high concentrations of exogenous ABA, and this method is believed to be saturated. To overcome this problem, a sensitized screen that used much lower concentration of exogenous ABA was performed to isolate new ABA insensitive mutants. Some of the isolated mutants had defects in light or retrograde signalling. One particular mutant (18-11) developed long hypocotyls under normal light condition. Based on its response to blue, red and far-red light conditions, this mutant is likely a novel hy mutant. Genetic analysis revealed that while ABA insensitivity in this mutant is recessive, the long hypocotyl phenotype is dominant. Positional cloning is currently being carried out to identify the gene. Findings from this study supports that ABA signalling interacts with light signalling networks.

The release of genetically modified, herbicide tolerant canola (<I>Brassica napus</i> L.) genotypes in western Canada has increased interest in the persistence of volunteer canola. <i>B. napus</i> seed may be induced into secondary dormancy in the laboratory, however, little is known of the seedbank ecology and the role of secondary dormancy as a persistence mechanism in this species in the region. The objectives of this research were i) to determine seedbank additions at the time of harvest, ii) determine the role of secondary seed dormancy in seedbank persistence under different management systems, iii) determine the relative importance of factors contributing to secondary dormancy potential and iv) evaluate the role of abscisic acid (ABA) during secondary dormancy induction in <I>B. napus</i>. On farms, average seedbank additions during harvest were approximately 20 times the normal seeding rate of canola. High secondary seed dormancy potential prolonged seedbank persistence in fields, irrespective of tillage system. <I>B. napus</i> exhibited seedling recruitment of a typical summer annual weed where seedling recruitment was only observed in the spring. Seasonal seedling recruitment was the result of two fates: seed death in the shallow seedbank, irrespective of dormancy potential, and increased ungerminability in buried seeds which was related to secondary seed dormancy potential. Among the factors that contribute to secondary seed dormancy potential, genotype was of greatest significance. Seed size was of lesser importance, while the contributions of pre-harvest factors including seed maturity, year, and location were negligible in comparison. Differences in ABA synthesis and the response to ABA application were related to secondary seed dormancy potential and correlated well previous reports linking ABA to seed dormancy. Conclusions that emerge from this research are i) that on some farms, seedbank additions may be lowered by more diligent harvest practices, ii) seedbank persistence of <I>B. napus</i> may be reduced by growing low dormancy genotypes and avoiding seed burial for one year after seedbank establishment and iii) ABA + ABA-glucose ester (ABA-GE) and the ability of seeds to respond to ABA application after seed dormancy induction may potentially be used to identify seed dormancy potential in this species.

The release of genetically modified, herbicide tolerant canola (<I>Brassica napus</i> L.) genotypes in western Canada has increased interest in the persistence of volunteer canola. <i>B. napus</i> seed may be induced into secondary dormancy in the laboratory, however, little is known of the seedbank ecology and the role of secondary dormancy as a persistence mechanism in this species in the region. The objectives of this research were i) to determine seedbank additions at the time of harvest, ii) determine the role of secondary seed dormancy in seedbank persistence under different management systems, iii) determine the relative importance of factors contributing to secondary dormancy potential and iv) evaluate the role of abscisic acid (ABA) during secondary dormancy induction in <I>B. napus</i>. On farms, average seedbank additions during harvest were approximately 20 times the normal seeding rate of canola. High secondary seed dormancy potential prolonged seedbank persistence in fields, irrespective of tillage system. <I>B. napus</i> exhibited seedling recruitment of a typical summer annual weed where seedling recruitment was only observed in the spring. Seasonal seedling recruitment was the result of two fates: seed death in the shallow seedbank, irrespective of dormancy potential, and increased ungerminability in buried seeds which was related to secondary seed dormancy potential. Among the factors that contribute to secondary seed dormancy potential, genotype was of greatest significance. Seed size was of lesser importance, while the contributions of pre-harvest factors including seed maturity, year, and location were negligible in comparison. Differences in ABA synthesis and the response to ABA application were related to secondary seed dormancy potential and correlated well previous reports linking ABA to seed dormancy. Conclusions that emerge from this research are i) that on some farms, seedbank additions may be lowered by more diligent harvest practices, ii) seedbank persistence of <I>B. napus</i> may be reduced by growing low dormancy genotypes and avoiding seed burial for one year after seedbank establishment and iii) ABA + ABA-glucose ester (ABA-GE) and the ability of seeds to respond to ABA application after seed dormancy induction may potentially be used to identify seed dormancy potential in this species.

Doctor of Philosophy / Horticulture, Forestry, and Recreation Resources / Sunghun Park / Abiotic stresses, including chilling and drought stresses, are considered to be major limiting factors for growth and yield of agricultural and horticultural crops. One of the inevitable consequences of abiotic stresses is the accumulation of reactive oxygen species (ROS) in plants. ROS can either act as an alarm signal to induce the defense pathway when kept at a low level or cause oxidative damage to various cellular components when increased to a phytotoxic level. Glutaredoxins (GRXs) are members of ROS scavenging system that can maintain the cell redox homeostasis by using the reducing power of glutathione. In this research, we characterized the roles of GRXs in protecting tomato (Solanum lycopersicum) from chilling stresses and rice (Oryza sativa L.) from drought stresses. Our results indicated that ectopic expression of an Arabidopsis gene AtGRXS17 in tomato could enhance the chilling tolerance by increasing antioxidant enzyme activities and reducing H₂O₂ accumulation to ameliorate oxidative damage to cell membranes and photosystems. Furthermore, AtGRXS17-expressing tomato plants had increased accumulation of soluble sugars to protect plant cells from dehydration stress. In rice, silenced expression of a rice glutaredoxin gene OsGRXS17 was used as a reverse-genetic approach to elucidate the roles of OsGRXS17 in drought stress tolerance. Our results showed that silenced expression of OsGRXS17 conferred improved tolerance to drought stress in rice. ABA-mediated stomatal closure is an important protection mechanism that plants adapt to a drought stress conditions, and H2O2 acts as secondary messenger in ABA signaling to induce the stomatal closure. Silenced expression of OsGRXS17 gave rise to H₂O₂ accumulation in the guard cells and promoted ABA-mediated stomatal closure, resulting in reduced water loss, higher relative water content, and consequently enhanced drought tolerance in rice. This research provides a new perspective on the functions of GRXs in chilling and drought stress tolerance of tomato and rice, and an important genetic engineering approach to improve chilling and drought stress tolerance for other crop species.

A banana é um fruto consumido no mundo todo e, ao contrário do que acontece na maioria dos países nos quais predomina o grupo Cavendish, uma grande variedade de cultivares são consumidas nas diferentes regiões do Brasil. No entanto, as informações bioquímicas e fisiológicas a respeito das cultivares consumidas no país são ainda restritas e, apesar de serem muito diferentes entre si, são tratadas da mesma maneira na pré e pós-colheita, o que muitas vezes compromete a qualidade do fruto. Diante disso, dois pontos são fundamentais para a obtenção de frutos de melhor qualidade: a definição do ponto de colheita e o tratamento dos frutos com etileno após a colheita. Hoje a colheita é feita em função do diâmetro dos frutos e o tratamento com etileno é o mesmo para todas as cultivares. Porém, frutos com o mesmo diâmetro nem sempre estão no mesmo estágio de maturidade fisiológica e, usualmente, o tratamento pós-colheita feito com etileno, visando o amadurecimento mais rápido e uniforme dos frutos, não segue nenhuma orientação técnica. A consequência da falta de critérios definidos para a colheita e para a aplicação de etileno resulta em bananas com baixa qualidade e com vida-de-prateleira curta. A produção de banana é dificultada pelos problemas fitossanitários que ocorrem nas plantações, incluindo doenças como as Sigatokas Negra e Amarela e o Mal-do-Panamá. Tendo em vista a ameaça que as doenças da bananeira representam e os prejuízos que podem causar, a introdução de cultivares resistentes é a melhor forma para reduzir a pressão desses patógenos sobre a cultura. Por ser resistente às Sigatokas e ao Mal de Panamá este trabalho visou conhecer melhor a cultivar Thap Maeo (Musa acuminata AAB cv. Thap Maeo) que tem como defeito principal uma vida-de-prateleira muito curta. Os objetivos deste trabalho foram: (1) estabelecer o ponto de colheita das bananas da cultivar Thap Maeo utilizando a metodologia da soma de temperatura a que a planta está exposta durante o desenvolvimento dos frutos; (2) estabelecer, a partir da caracterização físico-química dos frutos, o teor ideal de etileno exógeno para promover o amadurecimento uniforme dos frutos e, (3) estudar o balanço hormonal no amadurecimento dos frutos. Em uma primeira etapa foi implantado um experimento de campo para determinar a Temperatura Base e a Idade Fisiológica Máxima para esta cultivar. Estes parâmetros são necessários para o cálculo da idade fisiológica e na determinação do ponto de colheita. Com o término desta etapa, foram realizadas colheitas de frutos em diferentes épocas do ano para confirmar a metodologia usada. Foi possível estabelecer uma metodologia para estimar o ponto de colheita dos frutos de acordo com a estação do ano em que estes se desenvolveram. Para estabelecer o teor ideal de etileno exógeno aplicado na pós-colheita, os frutos foram testados com diferentes concentrações de etileno (0 a 1000 ppm) em dose única. Os resultados indicam que mesmo as menores concentrações de etileno aplicadas promovem o amadurecimento uniforme dos frutos, sem diferenças aparentes entre os tratamentos. Além disso, as análises do perfil de compostos voláteis da polpa indicam que a maior diferença entre o aroma dos tratamentos é entre frutos tratados e não tratados. Os frutos que não foram tratados apresentaram maior quantidade de compostos identificados. Em todas as colheitas realizadas, o período entre o início do amadurecimento e o amadurecimento completo dos frutos foi consideravelmente curto se comparado a bananas do subgrupo Nanica. Para melhor compreender estes resultados, além do etileno, foram quantificados os teores livres dos hormônios ácido indol-3-acético (AIA) e ácido abcísico (ABA). Em comparação com estudos anteriores, com cultivares do subgrupo Nanica, a cultivar Thap Maeo produz mais etileno e ABA durante o amadurecimento, além de apresentar menores teores de AIA quando verde. Estas diferenças hormonais podem explicar o curto período de amadurecimento desta cultivar. / Banana is an important staple food. Unlike the world Market, which has only the Cavendish Bananas as export bananas, Brazilian market has many Bananas varieties. However, biochemical and physiological information about these varieties are still scarce and different bananas cultivars are treated the same way in pre or post-harvest, which often affects the quality of the fruit. In this context, two issues are important to reach best fruit quality: definition of harvest time and the exogenous ethylene treatment. Now a day the harvest time is defined by the diameter of the fruits and the ethylene treatment is equal for all cultivars. However, fruits with the same diameter not always are in the same development grade and the post-harvest ethylene treatment, aiming a faster and uniform ripening, does not follow any technical recommendation. The consequences for the lack of criteria in the harvest and for the ethylene treatment might be a low quality and shelf life banana. Banana production is hampered by plant health problems occurring in the production fields, including the diseases such as the Sigatokas and the Panama Wilt. Given the threat of the banana diseases, and the damage they can cause, the introduction of resistant cultivars is the best way to reduce the pressure of these pathogens on this crop. Being resistant to Sigatokas and Panama diseases, this work aimed to know better the cultivar Thap Maeo (Musa acuminata AAB cv. Thap Maeo) whose main defect is a short shelf life. The objectives of this work were: (1) to establish the harvest time of Thap Maeo bananas using the thermal sum techniques, (2) using the physico-chemical characterization of the fruit, establish the ideal concentration of exogenous ethylene to promote uniform ripening of fruits and (3) study the hormonal balance in the fruit ripening. The first step was a field experiment to determine the base temperature and the maximum physiological age. These parameters are used to calculate the thermal sum. The next step was to harvest fruits from different times of the year to confirm the methodology. It was established a methodology to estimate the harvest time according to the season in which the fruit has developed. Five ethylene concentration were tested (0-1000 ppm). Results showed that even the low ethylene concentration applied could promote the uniform ripening of the fruits with no apparent differences between the treatments. Furthermore, the analysis of volatile compounds in the pulp indicate that the major difference between the aroma of treatments is between treated and untreated fruits. The fruits that were not treated showed a higher amount of identified compounds. For all harvested fruits, the period between the ripening start and full ripening of the fruit was short when compared to the Nanica bananas. Levels of the hormones indole-3-acetic acid and abscisic acid, in addition to ethylene, were quantified to better understand these results. Compared to previous studies, with cultivars of Nanica subgroup, the cultivar Thap Maeo produce more ethylene and ABA during ripening, and have lower IAA level in the green stage. These hormonal differences may explain the short maturity period of this cultivar.

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. Because NAE levels in seeds decline during imbibition similar to ABA, a physiological role was predicted for these metabolites in Arabidopsis thaliana seed germination and seedling development. There is also a corresponding increase of AtFAAH (fatty acid amide hydrolase), transcript levels and activity, which metabolizes NAE to ethanolamine and free fatty acids. Based on whole genome microarray studies it was determined that a number of up-regulated genes that were responsive to NAE were also ABA responsive. NAE induced gene expression in these ABA responsive genes without elevating endogenous levels of ABA. It was also determined that many of these NAE/ABA responsive genes were associated with an ABA induced secondary growth arrest, including ABI3. ABI3 is a transcription factor that regulates the transition from embryo to seedling growth, the analysis of transcript levels in NAE treated seedlings revealed a dose dependent, inverse relationship between ABI3 transcript levels and growth, high ABI3 transcript levels were associated with growth inhibition. Similar to ABA, NAE negatively regulated seedling growth within a narrow window of early seedling establishment. When seedlings are exposed to NAE or ABA within the window of sensitivity, the induction of genes normally associated with the ungerminated desiccation tolerant state resumed. The NAE tolerant FAAH overexpressor and the NAE sensitive FAAH knockout both had a NAE/ABA sensitive window similar to the wild type A. thaliana. The abi3-1 ABA insensitive mutant does not undergo growth arrest upon exposure to ABA, but NAE did induce growth arrest when treated within the sensitivity window. This evidence showed that although NAE functions within an ABA dependent pathway, it also functions in an ABA independent signaling pathway. The FAAH overexpressor is tolerant to NAE through its ability to quickly metabolize NAE from the growth media, yet it is hypersensitive to ABA. The FAAH overexpressor also displayed hypersensitivity to GA, which improved its delayed germination in non-stratified seed, while the FAAH knock out showed GA insensitivity. Overall, these results showed that NAE functions as a negative regulator of germinating seed and seedling growth in ABA dependent and independent signaling pathways, and that altered NAE metabolism may interfere with ABA/GA perception in germinating seed.

Plants rely on diverse strategies to regulate water loss during drought. The phytohormone abscisic acid (ABA) is a critical mediator of stomatal closure during water stress in seed plants. Studies in conifers identified diverging strategies in long-term drought of ABA-mediated dynamics, particularly a peaking type dynamic during long term drought in some conifers. Few studies have reported this dynamic in angiosperms, and no study has revealed the mechanism driving declines in ABA levels as drought progresses in peaking type species. To understand peaking type dynamics, we exposed the model peaking type gymnosperm species <i>Callitris rhomboidea</i> and the highly drought resistant evergreen angiosperm <i>Umbellularia californica</i> to controlled long-term drought. We measured leaf water potentials (Ψ_l), stomatal conductance, ABA and the ABA catabolite phaseic acid (PA) levels in potted plants during a prolonged but non-fatal drought. We aimed to determine which of three potential drivers of peaking type dynamic were responsible for this response: (1) increased catabolism of ABA into PA at a threshold Ψ_l , (2) ABA export from the leaf is enhanced under drought, and (3) ABA biosynthesis ceases at a threshold Ψ_l. During long term drought, the evergreen angiosperm species <i>U. californica</i> demonstrated peaking type ABA dynamics like gymnosperms. In both species, PA levels did not increase significantly, in fact, PA levels tracked ABA levels, suggesting that ABA catabolism to PA may be a function of ABA levels. Girdling experiments to determine whether export from the leaf drove declines in ABA levels demonstrated that of the majority of ABA was likely converted to ABA glucose ester (ABA-GE), an inactive storage form of ABA, and exported from shoots during drought. Finally, by rapidly dehydrating branched collected at different timepoints during long-term drought we were able to determine that ABA biosynthesis is completely down regulated in leaves that have been dehydrated beyond leaf turgor loss point. The decline in ABA levels in peaking type species appears conserved across seed plants and is mediated by high export rates in the form of ABA-GE. Future work should assess a more diverse selection of species as well as study long-term drought in less tolerant species to test whether ABA biosynthesis is deactivated in all species once Ψ_ldeclines below turgor loss point.

Orientador: Gustavo Habermann / Resumo: O alumínio (Al) é o metal mais abundante na crosta terrestre. Em solos ácidos (pH < 5,0) é encontrado principalmente na forma de Al3+, tóxico à maioria das plantas. Nas plantas sensíveis a esse metal, o primeiro e mais conspícuo sintoma de toxicidade é a inibição do crescimento das raízes, onde a maior parte do Al é covalentemente retido. Além disso, o Al também causa sintomas indiretos, reduzindo o crescimento da parte aérea. Tal redução tem sido associada à assimilação de CO2 (A), e evidências sugerem o comprometimento das reações fotoquímicas, além da redução de 30 a 80% da condutância estomática (gs). Além disso, o Al também tem sido associado à redução na hidratação do mesofilo, como ocorre em condições de limitação hídrica. Baseado nestes fatos, testamos a hipótese de que parte dos sintomas de fitotoxicidade ao Al em plantas sensíveis, sobretudo aqueles relacionados ao baixo crescimento e desenvolvimento da parte aérea, bem como menos trocas gasosas, ocorrem em resposta à ativação de mecanismos associados à percepção de deficiência hídrica, principalmente à biossíntese de ácido abscísico (ABA). Utilizamos duas espécies sensíveis ao Al, mas cujos sintomas de toxicidade são induzidos por concentrações significativamente distintas, como Solanum lycopersicum (100 M Al) e Citrus limonia (1480 M Al). As plantas foram cultivadas em solução de nutrientes com e sem Al, em experimentos independentes com duração de 10 e 90 dias, para S. lycopersicum e C. limonia, respectivamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Aluminum (Al) is the most abundant metal in the Earth's crust. In acidic soils (pH < 5.0) it is found mostly as Al3+, which is toxic to most plants. In Al-sensitive plants, the first and most conspicuous symptom is the inhibition of the growth of the root system, where most of Al is covalently retained. Aluminum also causes indirect symptoms, such as reduced shoot growth. In Al-sensitive plants, Al-reduced CO2 assimilation rate (A) is observed, and there is evidence suggesting the impairment of photochemical reactions, as well as 30 to 80% reduction in stomatal conductance (gs). In addition, Al has also been associated with a reduction in mesophyll hydration, as occurs under water-limited conditions. Based on these facts, we tested the hypothesis that part of Al symptoms in sensitive plants, especially those related to low shoot growth and development, as well as low gas exchange, occur in response to activation of mechanisms closely associated with the perception of water deficiency, especially as signalled by abscisic acid (ABA) biosynthesis. We used two Al-sensitive species, whose symptoms of toxicity are induced by significantly different concentrations, such as Solanum lycopersicum (100 M Al) and Citrus limonia (1480 M Al). Plants were grown in nutrient solution with and without Al, in independent experiments of 10 and 90 days, for S. lycopersicum and C. limonia, respectively. In the experiments with both species, biometric parameters, root system morphology, biomass, ... (Complete abstract click electronic access below) / Doutor

Seed germination during unhospitable environmental conditions can be a major barrier to direct seeding efforts in dryland systems. In the sagebrush steppe, Artemisia tridentata Nutt. ssp. wyomingensis and Artemisia arbuscula are important shrub species that are being used in restoration, but seeding success is highly sporadic due to inter-annual and intra-seasonal weather variability. Altering and expanding the period of germination, as a form of bet-hedging, may improve plant establishment. Our objective was to determine if we could expand the period of germination using plant growth regulators (PGRs) applied in a conglomerated seed coating treatment. In a laboratory study, the seed was either left untreated, conglomerated separately with two concentrations of a germination inhibitor, abscisic acid (ABA), or with two different germination promoters, gibberellic acid (GA3) and 1-Aminocyclopropane carboxylic acid (ACC), a precursor to ethylene. Seeds were incubated in a loam soil at five constant temperatures (5-25 C) for approximately three months. Results indicate that seed treatments with PGRs can delay or speed germination. The greatest response to the seed treatments was observed at 5 C. For example, at this temperature PGRs delayed the time for 25% of the seeds to germinate by a maximum of 35 and 21 d and decreased this time by 5 and 25 d for A. t. ssp. Wyomingensis and A. arbuscula, respectively. Field studies are needed to determine if the bet-hedging strategy developed in this study will increase the likelihood that some seeds will germinate during periods that are more favorable for plant establishment.

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