Genetic analysis of leaf rolling in wheat

Dissertação de mestrado em Química Farmacêutica Industrial, apresentada à Faculdade de Farmácia da Universidade de CoimbraAtualmente, as doenças neurodegenerativas como a isquemia cerebral, Alzheimer e Parkinson atacam em massa a população mundial. O acréscimo destas patologias crê-se estar relacionado com falhas na homeostase de zinco e de espécies reativas de oxigénio (ROS) no sistema nervoso central. Neste trabalho questões desta natureza foram investigadas nas sinapses das fibras musgosas da área CA3 do hipocampo de rato. Os estudos foram efetuados em fatias cerebrais utilizando sondas fluorescentes de zinco e de espécies reativas de oxigénio. Sinais intracelulares de zinco foram medidos utilizando o indicador Newport Green na forma permeante, que não complexa o zinco presente nas vesículas sinápticas e que é co-libertado com o glutamato. As variações de zinco foram induzidas por meio de despolarizações com KCl ou usando um meio extracelular com TEA e rico em cálcio. No primeiro caso verificou-se um aumento significativo dos sinais de fluorescência de zinco. Os resultados obtidos usando antagonistas de recetores de glutamato e um bloqueador de canais de cálcio dependentes do potencial, indicam que aqueles sinais têm origem na zona pós-sináptica. No meio com TEA e muito cálcio, usado para induzir quimicamente a potenciação de longa duração, que se considera representar uma forma de memorização ao nível celular, verificou-se uma diminuição reversível da intensidade de fluorescência. Este facto pode ser devido à activação, pelo zinco libertado, de canais de K/ATP pré-sinápticos, o que origina a hiperpolarização da membrana e uma diminuição na libertação de zinco. Alterações na formação de espécies reativas de oxigénio foram estudadas, por meio da sonda H2DCFDA permeante, que é sensível essencialmente aos radicais hidroxilo e peróxido de hidrogénio. Os resultados obtidos, induzidos de forma semelhante aos de zinco, têm dum modo geral um comportamento idêntico ao destes sinais Inúmeros avanços na indústria e na medicina prometem melhorias ao nível das patologias anteriormente mencionadas e outras. No entanto, existe o reverso da moeda, o aumento da industrialização e de fármacos disponíveis provoca um aumento da poluição em efluentes e águas residuais, tendo em conta que os processos usados nas estações de tratamento não são totalmente eficazes. III O sulfametoxazole (SMX) é um antibiótico largamente utilizado, que não é totalmente metabolizado pelo organismo, tendo por isso vindo a ser detetado em águas residuais. Os sinais de zinco medidos aumentam quando as fatias são expostas a esta sulfonamida, voltando ao nível inicial após a mudança para o meio extracelular normal. O efeito do sulfametoxazole nas espécies reativas de oxigénio também foi testado, tendo-se verificado um aumento daquelas espécies na presença de SMX. Os resultados mostram que a ação deste antibiótico não é reversível, por este motivo, as quantidades ambientais de SMX nas águas residuais assumem uma preocupação crescente. A observação de efeitos irreversíveis na formação de espécies reactivas de oxigénio durante actividade neuronal intensa, sugere que aquele fármaco pode contribuir para diversas patologias neurodegenerativas.Currently, neurodegenerative diseases such as cerebral ischemia, Alzheimer's and Parkinson's mass attack the world population. The increase of these diseases is believed to be related to failures in the homeostasis of zinc and of reactive oxygen species in the central nervous system. In this work such issues have been investigated in the mossy fiber synapses from area CA3 of the rat hippocampus. The studies were performed in brain slices using fluorescent probes of zinc and of reactive oxygen species. Intracellular zinc signals were measured using the indicator Newport Green in the permeant form, that does not complex the zinc present in the synaptic vesicles which is co-released with glutamate. The zinc changes were induced by KCl depolarization or applying an extracellular medium containing TEA and rich in calcium. In the first case a significant increase of the fluorescence zinc signals was observed. The results obtained using antagonists of glutamate receptors and one blocker of voltage-dependent calcium channels, indicate that those signals have a postsynaptic origin. In the medium containing TEA and high calcium, used to chemically induce long-term potentiation, which is considered to represent a way of memory formation at the cellular level, a reversible decrease of the fluorescence intensity was observed. This fact may be due to the activation, by released zinc, of presynaptic K / ATP channels, which leads to membrane hyperpolarization and to a decrease in zinc release. Changes in the formation of reactive oxygen species were studied by means of the permeant H2DCFDA probe, which is specially sensitive to the hydroxyl and hydrogen peroxide radicals. The results, induced in a similar way as for zinc, have in general an identical behaviour to that of these signals. Numerous advances in industry and medicine promise improvements in the above mentioned pathologies and others. However, there is the reverse of the coin, the increasing industrialization and availability of drugs results in increased pollution in waste waters and effluents, since the processes used in water treatment plants are not entirely effective. V Sulfamethoxazole (SMX) is a widely used antibiotic, which is not fully metabolized by the organism and, for this reason, is being detected in wastewaters. The measured zinc signals increase when the slices are exposed to this sulfonamide, returning to the initial level after changing to the normal extracellular medium. The effect of sulfamethoxazole in reactive oxygen species was also tested, having been observed an increase of those species in the presence of SMX. The results show that the action of this antibiotic is not reversible, therefore, their environmental quantities in wastewaters are of growing concern. The observation of irreversible effects on the formation of reactive oxygen species during intense neuronal activity, suggests that that pharmaceutical agent may contribute to various neurodegenerative pathologie

Evolution of leaf-form in land plants linked to atmospheric CO2 decline in the Late Palaeozoic era

The widespread appearance of megaphyll leaves, with their branched veins and planate form, did not occur until the close of the Devonian period at about 360 Myr ago. This happened about 40 Myr after simple leafless vascular plants first colonized the land in the Late Silurian/Early Devonian, but the reason for the slow emergence of this common feature of present-day plants is presently unresolved. Here we show, in a series of quantitative analyses using fossil leaf characters and biophysical principles, that the delay was causally linked with a 90% drop in atmospheric pCO2 during the Late Palaeozoic era. In contrast to simulations for a typical Early Devonian land plant, possessing few stomata on leafless stems, those for a planate leaf with the same stomatal characteristics indicate that it would have suffered lethal overheating, because of greater interception of solar energy and low transpiration. When planate leaves first appeared in the Late Devonian and subsequently diversified in the Carboniferous period, they possessed substantially higher stomatal densities. This observation is consistent with the effects of the pCO2 on stomatal development and suggests that the evolution of planate leaves could only have occurred after an increase in stomatal density, allowing higher transpiration rates that were sufficient to maintain cool and viable leaf temperatures

Water relations of evergreen and drought-deciduous trees along a seasonally dry tropical forest chronosequence

Seasonally dry tropical forests (SDTF) are characterized by pronounced seasonality in rainfall, and as a result trees in these forests must endure seasonal variation in soil water availability. Furthermore, SDTF on the northern Yucatan Peninsula, Mexico, have a legacy of disturbances, thereby creating a patchy mosaic of different seral stages undergoing secondary succession. We examined the water status of six canopy tree species, representing contrasting leaf phenology (evergreen vs. drought-deciduous) at three seral stages along a fire chronosequence in order to better understand strategies that trees use to overcome seasonal water limitations. The early-seral forest was characterized by high soil water evaporation and low soil moisture, and consequently early-seral trees exhibited lower midday bulk leaf water potentials (ΨL) relative to late-seral trees (−1.01 ± 0.14 and −0.54 ± 0.07 MPa, respectively). Although ΨL did not differ between evergreen and drought-deciduous trees, results from stable isotope analyses indicated different strategies to overcome seasonal water limitations. Differences were especially pronounced in the early-seral stage where evergreen trees had significantly lower xylem water δ18O values relative to drought-deciduous trees (−2.6 ± 0.5 and 0.3 ± 0.6‰, respectively), indicating evergreen species used deeper sources of water. In contrast, drought-deciduous trees showed greater enrichment of foliar 18O (∆18Ol) and 13C, suggesting lower stomatal conductance and greater water-use efficiency. Thus, the rapid development of deep roots appears to be an important strategy enabling evergreen species to overcome seasonal water limitation, whereas, in addition to losing a portion of their leaves, drought-deciduous trees minimize water loss from remaining leaves during the dry season

Carbon stable isotope analysis of cereal remains as a way to reconstruct water availability: preliminary results

Reconstructing past water availability, both as rainfall and irrigation, is important to answer questions about the way society reacts to climate and its changes and the role of irrigation in the development of social complexity. Carbon stable isotope analysis of archaeobotanical remains is a potentially valuable method for reconstructing water availability. To further define the relationship between water availability and plant carbon isotope composition and to set up baseline values for the Southern Levant, grains of experimentally grown barley and sorghum were studied. The cereal crops were grown at three stations under five different irrigation regimes in Jordan. Results indicate that a positive but weak relationship exists between irrigation regime and total water input of barley grains, but no relationship was found for sorghum. The relationship for barley is site-specific and inter-annual variation was present at Deir ‘Alla, but not at Ramtha and Khirbet as-Samra

Biotic carbon feedbacks in a materially-closed soil-vegetation-atmosphere system

The magnitude and direction of the coupled feedbacks between the biotic and abiotic components of the terrestrial carbon cycle is a major source of uncertainty in coupled climate–carbon-cycle models1, 2, 3. Materially closed, energetically open biological systems continuously and simultaneously allow the two-way feedback loop between the biotic and abiotic components to take place4, 5, 6, 7, but so far have not been used to their full potential in ecological research, owing to the challenge of achieving sustainable model systems6, 7. We show that using materially closed soil–vegetation–atmosphere systems with pro rata carbon amounts for the main terrestrial carbon pools enables the establishment of conditions that balance plant carbon assimilation, and autotrophic and heterotrophic respiration fluxes over periods suitable to investigate short-term biotic carbon feedbacks. Using this approach, we tested an alternative way of assessing the impact of increased CO2 and temperature on biotic carbon feedbacks. The results show that without nutrient and water limitations, the short-term biotic responses could potentially buffer a temperature increase of 2.3 °C without significant positive feedbacks to atmospheric CO2. We argue that such closed-system research represents an important test-bed platform for model validation and parameterization of plant and soil biotic responses to environmental changes

Stable isotope analysis provides new information on winter habitat use of declining avian migrants that is relevant to their conservation

Winter habitat use and the magnitude of migratory connectivity are important parameters when assessing drivers of the marked declines in avian migrants. Such information is unavailable for most species. We use a stable isotope approach to assess these factors for three declining African-Eurasian migrants whose winter ecology is poorly known: wood warbler Phylloscopus sibilatrix, house martin Delichon urbicum and common swift Apus apus. Spatially segregated breeding wood warbler populations (sampled across a 800 km transect), house martins and common swifts (sampled across a 3,500 km transect) exhibited statistically identical intra-specific carbon and nitrogen isotope ratios in winter grown feathers. Such patterns are compatible with a high degree of migratory connectivity, but could arise if species use isotopically similar resources at different locations. Wood warbler carbon isotope ratios are more depleted than typical for African-Eurasian migrants and are compatible with use of moist lowland forest. The very limited variance in these ratios indicates specialisation on isotopically restricted resources, which may drive the similarity in wood warbler populations' stable isotope ratios and increase susceptibility to environmental change within its wintering grounds. House martins were previously considered to primarily use moist montane forest during the winter, but this seems unlikely given the enriched nature of their carbon isotope ratios. House martins use a narrower isotopic range of resources than the common swift, indicative of increased specialisation or a relatively limited wintering range; both factors could increase house martins' vulnerability to environmental change. The marked variance in isotope ratios within each common swift population contributes to the lack of population specific signatures and indicates that the species is less vulnerable to environmental change in sub-Saharan Africa than our other focal species. Our findings demonstrate how stable isotope research can contribute to understanding avian migrants' winter ecology and conservation status

Water isotopes in desiccating lichens

The stable isotopic composition of water is routinely used as a tracer to study water exchange processes in vascular plants and ecosystems. To date, no study has focussed on isotope processes in non-vascular, poikilohydric organisms such as lichens and bryophytes. To understand basic isotope exchange processes of non-vascular plants, thallus water isotopic composition was studied in various green-algal lichens exposed to desiccation. The study indicates that lichens equilibrate with the isotopic composition of surrounding water vapour. A model was developed as a proof of concept that accounts for the specific water relations of these poikilohydric organisms. The approach incorporates first their variable thallus water potential and second a compartmentation of the thallus water into two isotopically distinct but connected water pools. Moreover, the results represent first steps towards the development of poikilohydric organisms as a recorder of ambient vapour isotopic composition