In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors. The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions. We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production. LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.

M.Sc. / Heat shock (HS) proteins and HS transcription factors (HSFs) have been coined as the ‘Achilles Heel’ for cancer therapy, since they have been found to be overexpressed in cancer cells and are required for cell survival during tumour progression and metastasis. Hsp70 and other members of the Hsp family have been shown to inhibit apoptosis at several different stages, contributing to resistance to chemotherapy. NSAIDs, like salicylates and aspirin, are used for the treatment and prevention of cancers such as breast cancer. SA has been shown to enhance HSF-DNA binding and results in the increased expression of heat-induced Hsp70 which is antiapoptotic. We hypothesise that SA treatment can result in the potentiation of Hsp70 in MCF-7 cells further increasing their resistance to apoptosis and thus the aim of this study was to investigate the dose-responsive effects of salicylic acid (SA) in the presence and absence of heat shock on components of the pro and antiapoptotic components of the apoptotic pathway. MCF-7 cells, which naturally overexpress Hsp70, were treated with several doses of SA in the presence and absence of a mild heat shock, followed by analysis of Hsp70 and several pro and antiapoptotic members of intrinsic and extrinsic apoptotic pathways, including Bcl-2, Bax, caspase 6 and 8, JNK, AIF and APAF-1. Induced Hsp70 accumulation by the SA treatments in the presence and absence of heat shock enhanced apoptosis in cells exposed to SA whereas higher concentrations of SA combination with heat shock induced necrosis and a decrease in Hsp70 accumulation in MCF-7 cells. Identification of the effects which specific concentrations of SA in the presence and absence of heat shock had on the apoptotic pathway constituents helped highlight potential pathways by which cell death could occur in MCF-7 cells through the downregulation of Hsp70. It is most likely that MCF-7 cell death is occurring due to the release of reactive oxygen species (ROS) which in turn lead to necrosis or death may be achieved via a cathepsin-B-mediated cell death pathway where both of these possibilities need to be further investigated.

Para representar o equilíbrio existem modelos termodinâmicos empíricos e semi-empíricos, porém nenhum deles tem aplicação generalizada. Muitos dos modelos utilizados para o equilíbrio sólido-líquido advêm dos modelos desenvolvidos para o equilíbrio líquido-vapor (ELV) e, em alguns casos, podem não representar adequadamente os sistemas reais, tornando-se mais críticos os desvios quando o sólido, ou o líquido, ou ambos, são polares. Para o estudo dos modelos e realizar as devidas comparações foram inicialmente realizados experimentos em laboratório com sistemas binários e ternários utilizando como solventes o etanol, a água e as suas misturas e como soluto o ácido salicílico, todas substâncias polares. Os dados experimentais foram obtidos utilizando uma variante do método isotérmico, tendo como sistemas binários: etanol-ácido salicílico e água-ácido salicílico e, como sistemas ternários as misturas dos solventes. Os experimentos envolveram a variação da concentração mássica de etanol em intervalos de 20%. A variação da temperatura foi feita em intervalos de 5°C de 20 a 55°C. A análise do total de 48 dados experimentais indicou que a solubilidade do ácido salicílico aumenta à medida em que a temperatura e/ou a concentração mássica de etanol no solvente aumenta. Esses dados foram utilizados no ajuste de diferentes modelos para ESL. Os modelos estudados foram: UNIFAC e GSP (de predição baseado no ELV), UNIQUAC, Wilson, NRTL (semi-empíricos de ajuste de parâmetros baseados no ELV), Nývlt e \'lâmbda\'h (semi-empíricos de ajuste de parâmetros baseados no ESL) e Margules (empírico de ajuste de parâmetros). Os resultados mostraram que os modelos de predição têm aplicação restrita quando aplicado ao sistema estudado. Entre os modelos de ajuste, o UNIQUAC resultou em menores desvios em relação aos dados experimentais, considerando-se toda a faixa de condições experimentais adotadas. Os resultados experimentais também foram utilizados no ajuste de um modelo baseado em rede neural, o qual foi utilizado para mapeamento do sistema e comparação com as previsões pelos modelos considerados. Simulações com rede neural resultaram em boa concordância com os resultados experimentais, indicando que tal modelo pode ser usado para prever solubilidade na faixa de condições do presente estudo. / The knowledge of solid-liquid equilibrium (SLE) is an important factor in crystallization and dissolution studies. In most of these systems equilibrium is represented by empirical and semi-empirical thermodynamical models, with no general application. Many models used in SLE result from models developed for vapor-liquid equilibrium (VLE) and in some cases these models do not represent real systems adequately, becoming critical when polar solids or liquids are present in the system. In the present work a study of thermodynamical models for SLE was carried out by comparing the performance of different models with experimental data of binary and ternary systems consisting of ethanol, water and their mixtures as solvents, and salicylic acid as solute. The experimental data were obtained using a variant of the isothermal method for the following binary systems: ethanol-salicylic acid and water-salicylic acid. In the study with ternary systems ethanol-water mixtures at different ratios were adopted as solvents, and salicylic acid was the solute. A total of 48 experiments were carried out by changing the ethanol mass fraction in the solvent from 0 to 100%, and the temperature from 20 to 55°C. The solubility of salicylic acid increases with the increase in the temperature and/or ethanol concentration in the solvent. The models considered in the study were: UNIFAC and GSP (prediction models based on vapor-liquid-equilibrium), UNIQUAC, Wilson and NRTL (semi-empirical models of fitted parameters based on vapor-liquid equilibrium), Nývlt and \'lâmbda\'h (semi-empirical models with fitted parameters based on SLE) and Margules (empirical model with fitted parameters). The results showed that the UNIQUAC model with fitted parameters can describe the SLE with reasonable accuracy, while all other methods resulted in poor agreement with the system\'s behavior, with systematic deviations from the experimental results. The system was also mapped with the use of a neural network model with parameters fitted to the experimental data. Simulation results with the neural network provided an accurate map of the system that can be used within the range of conditions considered in this study.

Viruses can affect plant-insect interactions by altering emission of plant volatile organic compounds (VOCs). Previous work in the lab suggested that VOCs emitted by tomato (Solanum lycopersicum) plants infected with cucumber mosaic virus (CMV) were more attractive to bumblebees (Bombus terrestris) in free choice assays. I extended this work using Arabidopsis thaliana mutants with lesions in genes encoding factors in RNA silencing. In conditioning assays, I confirmed that plant VOC emission is controlled in part by the microRNA regulatory pathway. I used gas chromatography coupled to mass spectrometry and principal component analysis to confirm that CMV infection caused changes in VOC emission by tomato. VOCs collected from non-flowering mock-inoculated and CMV-infected plants were qualitatively distinct from each other. CMV-infected plants also released greater quantities of VOCs than mock-inoculated plants. CMV appears to be both ‘turning up the volume’ of plant volatile emission, whilst ‘tuning’ volatile blend composition so as to diminish levels of a repellant signal. These data are likely to explain how bumblebees can discriminate between VOCs emitted by mock-inoculated and CMV-infected plants. To determine if CMV infection of tomato plants affects plant reproductive success, I carried out a series of bumblebee pollination experiments. Bumblebees pollinate tomato by ‘buzzing’ (sonicating) the flowers, which releases pollen and enhances self-fertilization and seed production as well as pollen export. First, I established that CMV-infected tomato plants produced fruits with a lower seed yield than mock-inoculated plants. When single bumblebees were allowed to buzz-pollinate flowers in a small flying arena, the fruit that developed from buzz-pollinated flowers on virus infected plants had significantly more seeds than fruit from non-visited flowers. Subsequent experiments were performed in a large flying arena. Bumblebees consistently spent longer foraging on the mock-inoculated tomato plants but seed yield was increased by bumblebee pollination in both mock-inoculated as well as virus-infected tomato plants. However, although buzz-pollination significantly enhanced seed yield from CMV-infected plants compared to fruit from non-buzz-pollinated flowers, the yield was higher in buzz-pollinated fruits from mock-inoculated plants. Similar experiments were carried out utilizing a transgenic line of tomato that constitutively expresses the green fluorescent protein in order to estimate the level of cross-pollination from either CMV-infected plants to mock-inoculated plants or vice versa. More pollen from virus-infected plants was transferred to mock-inoculated plants than the reciprocal cross. However, some caution is needed in the interpretation of the larger scale experiments because the tomato plants were affected by a fungal infection. I investigated if the defensive plant hormone salicylic acid (SA) affects bee-perceivable VOC emission. Exogenous SA treatment renders non-flowering tomato plants more attractive to bumblebees in free choice experiments in which bees could only perceive VOCs, but bumblebees spent less time visiting SA-treated flowering tomato plants in the glasshouse (when the bumblebees were allowed unimpeded access to the flowers). Taken together, these data provide evidence that virus infection can affect host-pollinator interactions. Speculatively, CMV infection may change the fitness of susceptible plants via changes in production of pollinator-attractive VOCs and this may affect the balance of resistant or susceptible plants within the host population.

Os comportamentos espectroeletroquímicos do ácido acetilsalicílico (AAS) e seu produto de hidrólise, o ácido salicílico (AS), foram estudados em soluções aquosas nas regiões de pH ácido, neutro e alcalino. Resultados para experimentos de voltametria cíclica sugeriram possíveis processos de eletro-oxidação e eletro-redução dos fármacos. O monitoramento do espectro de absorbância na região do UV-vis, simultâneo à medida de carga envolvida na eletrólise, permitiu a identificação de processos redox e o cálculo do número de elétrons envolvidos, aplicando a Lei de Faraday. O fármaco mostrou-se estável em pH ácido, reduziu em pH neutro e oxidou em pH alcalino. Tanto no processo de eletro-redução como na eletro-oxidação, os mecanismos propostos estabelecem o envolvimento de 1 elétron para a identificação de mudanças no nível molecular. Estas foram observadas pelas alterações de espectros de absorbância na região do UV-vis. Técnicas complementares, ressonância paramagnética eletrônica (RPE) e espectroscopia de transmitância FT-IR, foram usadas para a caracterização dos produtos obtidos em experimentos de eletrólise. As respostas espectrofotométricas associadas à processos eletroquímicos permitiram o desenvolvimento de método espectroeletroquímico para a detecção do fármaco em amostras reais contidas em soluções com pH neutro, utilizando a técnica de voltabsormetria derivada linear (DLVA). A interação entre os fármacos e íons de ferro no ambiente do estômago, foi simulada em experimentos in vitro, empregando eletroquímica e espectrofotometria. Na presença do AAS ocorreram interações fracas sem a interferência para a absorção de ferro pelo organismo. Em contrapartida, o AS interagiu formando um complexo estável com o Fe3+, podendo ser apontado como um potencial interferente para a absorção de ferro provocando anemia em indivíduos vegetarianos que fazem uso contínuo deste fármaco. / The spectroelectrochemical behavior of acetylsalicylic acid (ASA) and its spin off hydrolysis, salicylic acid (SA), were studied in aqueous solutions in the acid, neutral and alkaline pH regions. Results for cyclic voltammetry experiments suggested a possible electro-oxidation and electro-reduction of the drugs. The monitoring of the absorbance spectra in the region of the UV-vis, simultaneously with the measurement of the charge involved in the electrolysis, allowed the identification of redox processes and the calculation of the number of electrons involved applying Faraday\'s Law. The drug was stable in acid solutions, reduced in neutral and oxidized in alkaline ones. In electro-reduction and electro-oxidation processes, the proposed mechanisms establish the involvement of 1 electron to identify changes at the molecular level. These were observed by changes in absorbance spectra in the UV-vis region. Complementary techniques, electronic paramagnetic resonance (EPR) and FT-IR transmittance spectroscopy were used to characterize the products obtained in electrolysis experiments. The spectrophotometric responses associated to the electrochemical processes allowed the development of a spectroelectrochemical method for the detection of the drug in real samples contained in solutions with neutral pH, using the technique of derivative linear voltabsorptometry (DLVA). The interaction between drugs and iron ions in the stomach environment was simulated in in vitro experiments using electrochemistry and spectrophotometry. In the presence of ASA, weak interactions occurred without interference for the absorption of iron by the organism. On the other hand, AS interacted to form a stable complex with Fe3+ and could be considered as a potential interfering agent for the iron absorption, causing anemia in vegetarian individuals who make continuous use of this drug.

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant known to accumulate less SA. Taken together expression of SBIP-428 is negatively correlated to the levels of SA in plants. The AtSRT2 plants exhibit no altered growth phenotype but exhibit a higher pathogen resistance against bacterial pathogen. Our results indicate that SBIP-428 is an important regulator in plant defense pathway.

Plants are in a perpetual evolutionary arms race with a wide range of pathogens. Their sessile nature has led plants to evolve defense mechanisms that can quickly recognize a unique stressor and deploy a resistance tailored for a specific attack. The salicylic acid (SA) mediated defense pathway has been shown to be one of the major defense tactics plants can initiate to defend themselves against microbial pathogens. Following a pathogen attack high levels of methyl salicylate (MeSA) are produced that can be converted to SA by the enzyme salicylic acid binding protein 2 (SABP2). A yeast two-hybrid screening was performed to identify protein interactions with SABP2 to better understand the regulation of the enzyme on a cellular level. SBIP-436 is an interacting protein of tobacco SABP2 which showed high homology to phospholipase D-δ (PLD- δ). With an abundance of stimulators PLD- δ may be a lipid signaling modulator developed to perform various functions in different situations. PLD- δ may be a key player in a lipid signaling cascade in the SA mediated defense pathway. We present a novel Nicotiana tabacum PLD- δ putative gene construct. We demonstrate that the putative PLD- δ is subject to alternative splicing and its expression is differentially modulated under biotic and abiotic stress. Our results indicate that this putative PLD- δ may play a role in the SA mediated defense pathway.

<p>In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors.</p><p>The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions.</p><p>We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production.</p><p>LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.</p>

Seasonally high nighttime temperatures (HNT) along the United States Gulf Coast and in regions of similar climate, during the critical stages of development, could reduce rice yield and quality. To study the effects of HNT on plant physiology, a method for applying a controlled heating treatment to plant canopies was developed using overhead infrared heaters, which are relatively inexpensive and are accurate, precise and reliable in rapidly controlling the temperature. The apparatus successfully maintained air temperatures within the set points plus/minus 0.5 degrees C, and was used for all the experiments. Several experiments were conducted to determine the response of various physiological parameters during and following exposure of rice plants to HNT (32 degrees C) or ambient nighttime temperature (ANT) (27 degrees C) starting from 2000 h until 0600 h, and with or without plant growth regulator treatments. The plant growth regulator treatments included alpha-tocopherol (vitamin E), glycine betaine (GB), and salicylic acid (SA), which play different roles in inducing thermo-tolerance in plants. High nighttime temperature had no effect on plant height, number of tillers and panicles, or rice net leaf photosynthetic rates. However, HNT increased leaf respiration (dark respiration in the night) (21%) and decreased membrane thermo-stability (60%), pollen germination (20%), spikelet fertility (18% as a % of total spikelets), grain length (2%), and grain width (2%). The HNT also hastened plant development. The combinations of these effects decreased rice yield by 90%. Moreover, under HNT, there were decreases in leaf chlorophyll concentration (7%) and nitrogen concentration (18%). Application of GB and SA increased total antioxidant capacity of the rice plants by 17%, thereby decreasing the leaf respiration rates, increasing membrane thermo-stability, pollen germination, and spikelet fertility, thus increasing the yield. High nighttime temperature decreased leaf starch concentration (14%), grain total nonstructural carbohydrate (TNC) concentration (9%), and grain extractable invertase activity (20%). Vitamin E- or GB-treated plants had greater grain soluble-sugar concentrations, whereas SA-treated plants had greater leaf soluble-sugar concentrations and lower grain TNC concentrations. Invertase activity was shown to be not rate limiting or required for sucrose degradation for starch synthesis in grain of 'Cocodrie' rice under short-term high nighttime temperatures exposures during grain filling. In conclusion, HNT decreased rice yield by increasing plant respiration, rate of crop development, and decreasing membrane thermo-stability, pollen germination, spikelet fertility and grain dimensions. Exogenous application of GB and SA increased yields under HNT, possibly acting through increased antioxidant levels, which might have protected the membranes and enzymes against heat-induced ROS-mediated degradation.

An efficient micropropagation protocol for large scale production of hazelnut plants is required for consistent supply of elite germplasm to support the growing industry in Ontario. The main focus of current research was to develop a bioreactor based micropropagation protocol for hazelnut multiplication. An integrated approach was developed to increase the multiplication rate by optimizing the nutrient medium supplements and culture technology using a temporary immersion bioreactor system (TIS). As compared to conventional semi-solid and liquid based culture system, the Liquid LabTM temporary immersion bioreactor system (LIS) showed a significant enhancement in the number of shoots (3.3 shoots/explant), shoot height, leaf area and chlorophyll content in micropropagated plantlets. Antioxidant supplements such as ascorbic acid and melatonin along with iron (460 µM FeEDDHA) significantly increased the shoot proliferation (5.5 shoots/explant). However, a much higher shoot proliferation (10.9 shoots/explant) was observed with the addition of aspirin (10 µM) in the presence of BA (17.6 µM). Among several hazelnut cultivars, HF-16 had the highest multiplication rate followed by Geneva and Epsilon. Medium supplemented with 10 µM IBA was the best for rooting of microshoots of HF-16 and the plantlets acclimatized in the green house with 80% survival rate. / Ontario Ministry of Agriculture and Food and Rural Affairs