The physiological responses to salt stress were measured in Chenopodium quinoa. In a greenhouse experiment, salt water was applied to the quinoa varieties, Chipaya and KU-2, and to the model halophyte Thellungiella halophila to assess their relative responses to salt stress. Height and weight data from a seven-week time course demonstrated that both cultivars exhibited greater tolerance to salt than T. halophila. In a growth chamber experiment, three quinoa cultivars, Chipaya, Ollague, and CICA 17 were hydroponically grown and physiological responses were measured with four salt treatments. Tissues collected from the growth chamber treatments were used to obtain leaf succulence data, tissue ion concentrations, compatible solute concentrations, and RNA for real-time PCR. Stomatal conductance and fresh weight were measured to determine the degree of stress and recovery. The expression profiles of SOS1, NHX1, and TIP2, genes involved in salt stress, showed constitutive expression in root tissue and up-regulation in leaf tissue in response to salt stress. These data suggest that quinoa tolerates salt through a combination of exclusion and accumulation mechanisms.

Made available in DSpace on 2015-03-26T13:51:49Z (GMT). No. of bitstreams: 1 texto completo.pdf: 243475 bytes, checksum: 08375cc332ea6491435cc3b67f4a34e9 (MD5) Previous issue date: 2010-03-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Programs of control and management of the quality in laboratories, the necessity of standardization of the analysis methods and repeatability of the experimental data have stimulated the increase of the use of Reference Material (RM) in the microbiological analyses. Considering that the stages of preparation and storage of the MR are stress processes that causes to injuries to the microorganism test the preparation of the cells becomes necessary to tolerate the variation of temperature and humidity the one that is submitted during the frezze- drying and storage. It was objectified, in this work, to get resistant cells of Salmonella enterica the frezze-drying for the RM preparation and to evaluate the stability of these cells throughout the stockage. The growth of enteric Salmonella sorotipo Enteritidis PT4 578 was folloied in broth brain heart infusion (BHI) and mineral salts medium (MMS) increased of 0,5 M sodium chloride (NaCl), 3 mM of trehalose e, or 0,05 mM of glucose. The survival after the frezze-drying was determined by the counting of the number of form units of colonies (UFC.g-1). The effect of the thermal shock in the cells of Salmonella Enteritidis PT4 578 was evaluated the 50 °C in the increase of the resistance to the frezze- drying. Also the influence of substrata of dehydration in the resistance of cells of Salmonella Enteritidis PT4 578 and the stability of the RM was evaluated during 90 days of stockage in the 4 °C the - 20 °C. The way of culture that guaranteed the biggest survival of the cells of Salmonella Enteritidis PT4 578 to the frezze-drying was the MMS, added of the evaluated solutos indicating it nutricional stress confers resistance crossed to the frezze-drying. The thermal shock diminishes the cellular viability, after the frezze-drying. The addition of 100 mM of trealose e, or sacarose to skimmed milk reconstituted (LDR) the 10%, increased the stability of the cells of Salmonella Enteritidis PT4 578 in the storaged MR the 4 °C and - 20 °C. The temperature of stockage of 4 °C resulted in loss of the cellular viability in about 2 logarithmic cycles after 90 days stockage while the -20 °C the loss in the cellular viability was ten times lesser. The results detach that, for the attainment of MR with bigger stability, it has the necessity of the preparation of the cells of Salmonella Enteritidis PT4 578 to present greater resistance the frezze-drying, considering conditions of nutricional stress and osmotic during the culture and the inclusion of osmoprotectant in the dehydration substratum. / Programas de controle e gestão da qualidade em laboratórios, a necessidade de padronização dos métodos de análise e repetibilidade dos dados experimentais têm impulsionado o aumento da utilização de Material de Referência (MR) nas análises microbiológicas. Considerando que as etapas de preparo e armazenamento do MR são processos estressantes que ocasionam injúrias ao micro-organismo teste faz-se necessário a preparação das células para tolerar a variação de temperatura e umidade a que são submetidas durante a liofilização e o armazenamento. Objetivouse, neste trabalho, obter células de Salmonella enterica resistentes a liofilização para o preparo de MR e avaliar a estabilidade destas células ao longo da estocagem. O crescimento de Salmonella enterica sorotipo Enteritidis PT4 578 foi acompanhado em caldo infusão cérebro coração (BHI) e meio mínimo de sais (MMS) acrescido dos solutos 0,5 M de cloreto de sódio (NaCl), 3 mM de trealose e, ou 0,05 mM de glicose. A sobrevivência após a liofilização foi determinada pela contagem do número de unidades formadoras de colônias (UFC.g-1). Avaliou-se o efeito do choque térmico nas células de Salmonella Enteritidis PT4 578 a 50 °C por 30 min no aumento da resistência à liofilização. Foi também avaliada a influência dos substratos de desidratação na resistência de células de Salmonella Enteritidis PT4 578 e a estabilidade do MR preparado durante 90 dias de estocagem a -20 °C e 4 °C. O meio de cultivo que garantiu a maior sobrevivência das células de Salmonella Enteritidis PT4 578 à liofilização foi o MMS, adicionado de NaCl, trealose e sacarose indicando que o estresse nutricional confere resistência cruzada à liofilização. O choque térmico diminui a viabilidade celular, após a liofilização. A adição de 100 mM de trealose e, ou sacarose ao leite desnatado reconstituído (LDR 10 %) como substrato de desidratação, aumentou a estabilidade das células de Salmonella Enteritidis PT4 578 no MR estocado a 4 °C e - 20 °C. A temperatura de estocagem de 4 °C resultou em perda da viabilidade celular em cerca de 2 ciclos logarítmicos após 90 dias de estocagem enquanto a -20 °C não houve perda na viabilidade celular significativa. Os resultados indicam que, para a obtenção de MR com maior estabilidade, há a necessidade do preparo da células de Salmonella Enteritidis PT4 578 para apresentar maior resistência a liofilização, submetendo estas células a condições de estresses nutricional e osmótico durante o cultivo e a inclusão de osmoprotetores (trealose ou sacarose) no substrato de desidratação.

Philosophiae Doctor - PhD / The role of phenolic acids in mitigating salt stress tolerance have been well documented. However, there are contradicting reports on the effect of exogenously applied phenolic acids on the growth and development of various plants species. A general trend was observed where phenolic acids were shown to inhibit plant growth and development, with the exception of a few documented cases. One of these such cases is presented in this thesis. This study investigates the role of exogenously applied p-coumaric acid (p-CA) on physio-biochemical and molecular responses of chia seedlings under salt stress. This study is divided into three parts. Part one (Chapter 3) focuses on the impact of exogenous p-coumaric acid on the growth and development of chia seedlings. In this section, chia seedlings were supplemented with exogenous p-CA and the various biochemical and plant growth parameters were measured. The results showed that exogenous p-CA enhanced the growth of chia seedlings. An increase in chlorophyll, proline and superoxide oxide contents were also observed in the p-CA treatment relative to the control. We suggested that the increase in chia seedling growth could possibly be via the activation of reactive oxygen species-signalling pathway involving O2− under the control of proline accumulation (Chapter 3). Given the allopathy, nature of p-coumaric acid it is noteworthy that the response observed in this study may be species dependent, as contrasting responses have been reported in other plant species. Part two (Chapter 4) of this study investigates the influence of piperonylic acid (an inhibitor of endogenous p-coumaric acid) on the growth and development of chia seedlings. In trying to illustrate whether p-CA does play a regulatory role in enhancing pseudocereal plant growth, we treated chia seedlings with the irreversible inhibitor of C4H enzyme, to inhibit the biosynthesis of endogenous p-CA. In this section, chia seedlings were treated with piperonylic acid and changes in plant growth, ROS-induced oxidative damage, p-CA content and antioxidant capacity was monitored. Inhibition of endogenous p-CA restricted chia seedling growth by enhancing ROS-induced oxidative damage as seen for increased levels of superoxide, hydrogen peroxide and the extent of lipid peroxidation. Although an increase in antioxidant activity was observed in response to piperonylic acid, this increase was not sufficient to scavenge the ROS molecules to prevent oxidative damage and ultimate cellular death manifested as reduced plant growth. The results presented in this section support our hypothesis that p-CA play an important regulatory role in enhancing chia seedling growth and development as shown in Chapter 3. Part three (Chapter 5) seeks to identify and functionally characterise p-coumaric acid induced putative protein biomarkers under salt stress conditions in chia seedlings. Previous studies have shown that p-CA reversing the negative effect caused by NaCl-induced salt stress. While these studies were able to demonstrate the involvement of p-CA in promoting plant growth under salt stress conditions, they focussed primarily on the physiological aspect, which lacks in-depth biochemical and molecular analysis (ionomic and proteomic data) which could help in detecting the genes/proteins involved in salt stress tolerance mechanisms. A comparative ionomics and proteomic study was conducted, with the aim of elucidating the pivotal roles of essential macro elements and/or key protein markers involved in p-CA induced salt stress tolerance in chia seedlings. With the exception of Na, all the other macro elements were decreased in the salt treatment. Contrary to what was observed for the salt treatment most of the macro elements were increased in the p-CA treatment. However, the addition of exogenous p-CA to salt stressed seedlings showed an increase in essential macro elements such as Mg and Ca which have been shown to play a key role in plant growth and development. In the proteomic analysis we identified 907 proteins associated with shoots across all treatments. Interestingly, only eight proteins were conserved amongst all treatments. A total of 79 proteins were unique to the p-CA, 26 to the combination treatment (NaCl + p-CA) and only two proteins were unique to the salt stress treatment. The unique proteins identified in each of the treatments were functionally characterised to various subcellular compartments and biological processes. Most of the positively identified proteins were localised to the chloroplast and plays key roles in photosynthesis, transportation, stress responses and signal transduction pathways. Moreover, the protein biomarkers identified in this study (especially in the p-CA treatment) are putative candidates for genetic improvement of salt stress tolerance in plants.