Copping with extreme dehydration:a physiological, biochemical and molecular study on the aquatic bryophyte Fontinalis antipyretica

Tese de doutoramento, Biologia (Fisiologia e Bioquímica), Universidade de Lisboa, Faculdade de Ciências, 2013Water is the most important element for life. During land invasion, the first plants had to face an extreme dry environment, undergoing desiccation, a process in which tissues virtually lose almost all water. Therefore, plants developed desiccation tolerance (DT) mechanisms through which they could experience the dry state and return to normal function upon rehydration. Nowadays, the organisms that have the ability to tolerate desiccation include members of different taxa such as microbes, lichens, bryophytes, vascular plants and animals. Nevertheless, DT is more frequent in lichens and bryophytes. Researchers have been closely studying DT mechanisms hoping to identify new genes that can be transferred through biotechnology to crop species, increasing their drought tolerance. In the Mediterranean region, the aquatic species Fontinalis antipyretica is periodically exposed to desiccation, in intermittent streams that lose their water during the dry season. Field observations suggest DT mechanisms in this aquatic bryophyte. The main objective of this Thesis is to study DT through the combination of physiological, biochemical and molecular techniques in a bryophyte from a habitat not usually reported as prone to desiccation, comparing with bryophytes already studied and described as desiccation tolerant. Moreover, this integrated study aims to identify DT processes/mechanisms that are common across desiccation tolerant plant species. Photosynthesis is a very sensitive indicator of desiccation. The measurement of the oxygen production rate, coupled with chlorophyll a fluorescence, allowed to follow the photosynthetic response through non-invasive techniques (Chapter 2). Examining the metabolic response to desiccation, we aimed to establish if F. antipyretica was DT and whether the recovery of this species was mainly determined by the extent or by the rate of dehydration, or by both. Our findings showed that the metabolic response of F. antipyretica to desiccation, both under field and laboratory conditions, is consistent with a DT pattern. It was concluded that dehydration must proceed slowly for the bryophyte to regain its predesiccation state following rehydration. This was further confirmed in field-desiccated samples which showed a similar recovery pattern as slow dehydration. This physiological study of a widely distributed aquatic bryophyte periodically subjected to desiccation contributed to improve our knowledge about dehydration rate in bryophyte survival. The metabolic impairment observed during dehydration led to an increase in reactive oxygen species (ROS) production (Chapter 3). It was found that managing oxidative stress is one critical aspect for surviving desiccation. Although ROS production in response to desiccation/rehydration has been determined in terrestrial bryophytes it was not investigated in aquatic bryophytes. In addition, there were no published studies examining the impact of dehydration rate on ROS production in bryophytes previously subjected to desiccation and no information on the impact of drying rate on its production. Since it was determined that dehydration rate is fundamental for surviving DT, we investigated whether this response included an oxidative burst sensitive to dehydration rate using an innovative approach combining ROS-specific probes and confocal microscopy. The response was a very high ROS production under fast dehydration whereas under slow dehydration was almost absent. ROS react with cellular constituents, such as protein and lipids, leading to damage and, thus, affecting directly cell survival. After observing the effect of dehydration rate on ROS production, the next step was to test if the drying rate affected membrane damage (Chapter 4). We measured lipid peroxidation as well as cation dynamics and conductivity measurements to assess membrane damage and permeability. Since NO as also been associated with DT and a possible role in membrane protection, we measure and discuss the possible roles of its production and action. We also found that the increase in ROS and red autofluorescence can be used in future works in desiccation and drought stress as an indicator for detecting membrane damage and cell rupture. During dehydration, cell water relations change occurring in some cases osmotic adjustment through sucrose accumulation. Sucrose has an important role in DT by preventing denaturation of macromolecules and slowing down damaging reactions with ROS. We investigated how contrasting (fast and slow) dehydration rates change cell water relations and sucrose content in an aquatic bryophyte and if those changes can explain the requirement of slow dehydration to induce DT (Chapter 5). The characteristics of the bryophytes cell walls appear to change under fast dehydration, allowing it to become more elastic which probably allows to maintain a functional metabolism to lower water potentials. Sucrose also increases under fast dehydration which can be an attempt to minimize damage when time for a more effective protection is not available. The theory for DT was based in a constitutive protection mechanism that would allow the bryophyte to tolerate and protect the structures during desiccation, coupled with a repairbased mechanism upon rehydration which would repair damage that accumulated during the dried state. This would be controlled at the transcriptional level by accumulation of mRNA that would be activated during rehydration. However, recently the study of proteomes in bryophytes in response to dehydration suggests another approach to DT. We investigated the effect of fast and slow drying rates on the protein profiles, considering both dehydration and rehydration (Chapter 6). After fast dehydration, the proteome profiles of F. antipyretica are very similar to control. However, rehydration following fast dehydration leads to loss of almost all proteins, providing evidence that the bryophyte does not have enough time to prepare for desiccation under this dehydration regime. Nevertheless, under slow dehydration there are substantial changes in the proteome profile, both during dehydration and rehydration which might indicate an induction of DT mechanisms under these circumstances. The development of desiccation tolerance mechanisms that involved the accumulation of specific proteins during cycles of dehydration/rehydration allowed land colonization by early bryophyte ancestors. The basis for such tolerance relies on common patterns of protein expression and metabolic adjustments which are very similar even in bryophytes from very distinct habitats. During dehydration, photosynthesis shuts down, high levels of soluble sugars occur in the cytoplasm, defence proteins increase, cytoskeleton is disassembled and sugar metabolism enzymes are up-regulated. After rehydration, photosynthesis restart, cytoskeleton is re-assembled, high levels of: soluble sugars, sugar metabolism enzymes and defence proteins are maintained. The main conclusion suggested by this work is that DT at the cellular level, namely at the level of the molecular mechanisms, is similar among bryophytes independently of their preferred habitat. Furthermore, it states that DT is induced by slow dehydration rate being eventually controlled to some point by the morphology, being the determinant factor in the adaptation of bryophytes to each habitat and desiccation conditions.A água é o elemento mais importante para a Vida na Terra. Durante a ocupação do meio terrestre, as primeiras plantas enfrentaram um ambiente extremamente árido levandoas à dessecação, um estado no qual os tecidos perdem praticamente toda a água. Desta forma, foram-se desenvolvendo mecanismos de tolerância à dessecação (DT, do inglês desiccation tolerance) nas plantas, graças aos quais estas podem submeter-se ao estado de dessecação e regressar a um funcionamento normal após a reidratação. Atualmente, os organismos que apresentam DT incluem membros de taxa muito distintos, tais como micróbios, briófitos, líquenes, plantas vasculares e animais. No entanto, a DT é mais frequente nos líquenes e nos briófitos. Os investigadores têm estudado os mecanismos de DT na esperança de descobrir novos genes que possam ser transferidos por meio da biotecnologia para espécies vegetais de interesse agrícola, aumentando sua tolerância à seca. Na região do Mediterrâneo, a espécie aquática de briófito Fontinalis antipyretica é periodicamente exposta à dessecação, em riachos que perdem a água durante a estação seca. Observações no campo sugerem mecanismos de DT neste briófito aquático. O objetivo principal desta Tese é estudar a DT através da combinação de técnicas fisiológicas, bioquímicas e moleculares num briófito proveniente de um habitat que normalmente não está sujeito à dessecação, comparando com briófitos já estudados e descritos como tolerantes à dessecação. Além disso, este estudo integrado pretende identificar processos/mecanismos de DT que sejam comuns nas diversas espécies vegetais tolerantes à dessecação. A fotossíntese é um indicador muito sensível à dessecação. A resposta fotossintética foi seguida por meio de técnicas não-invasivas através da medição da taxa de produção de oxigénio, acoplado com a fluorescência da clorofila a (Capítulo 2), do briófito F. antipyretica de modo a determinar o grau de DT. Examinou-se a resposta metabólica à dessecação e de que forma ocorre a recuperação, se é determinada principalmente pela extensão ou a taxa de desidratação, ou por ambas. Os nossos dados mostraram que a resposta metabólica de F. antipyretica à dessecação, tanto sob condições de campo como em laboratório, é consistente com um padrão de DT. No entanto, a desidratação deve desenrolar-se lentamente para o briófito recuperar totalmente o seu estado inicial após reidratação. Isto foi confirmado em amostras dessecadas no campo as quais possuem um padrão de recuperação semelhante ao das amostras desidratadas lentamente. Este estudo fisiológico de um briófito aquático com distribuição global contribuiu para melhorar o nosso conhecimento sobre o papel desempenhado pela taxa de dessecação na sobrevivência dos briófitos.Durante a desidratação, o normal funcionamento metabólico fica comprometido, originando um aumento do stresse oxidativo, especialmente na produção de espécies reativas de oxigénio (ROS, do inglês reactive oxygen species) (Capítulo 3). Desta forma, a manutenção de níveis aceitáveis de stresse oxidativo que permitam o funcionamento celular é um aspeto crítico na sobrevivência à dessecação. Embora a produção de ROS em resposta à dessecação / reidratação tenha sido observada em briófitos terrestres, o mesmo não sucedeu em briófitos aquáticos. Além disso, não existiam estudos publicados que relacionassem o impacto da taxa de desidratação na produção de ROS em briófitos previamente submetidos a dessecação. Uma vez que a taxa de desidratação é fundamental para sobreviver à dessecação, investigámos se essa resposta incluiu um burst oxidativo sensível a taxa de desidratação utilizando uma abordagem inovadora que combina sondas específicas para ROS e microscopia confocal. Após reidratação, observou-se uma elevada produção de ROS em amostras desidratadas rapidamente, enquanto nas desidratadas lentamente essa produção era quase ausente. As ROS reagem com componentes celulares, tais como proteínas e lípidos, levando a danos celulares, afetando, desta forma, a sobrevivência celular. Depois de observar o efeito da velocidade de desidratação na produção de ROS, o passo seguinte foi testar como a taxa de desidratação afetava a estrutura e a estabilidade membranares (Capítulo 4). Através da quantificação de peroxidação lipídica, da dinâmica de catiões e das medições de condutividade, foram avaliados os danos e permeabilidade membranares. Dado que o óxido nítrico também foi associado com DT, tendo um possível papel na proteção da membrana, procedemos à sua quantificação e discutimos os possíveis papéis da sua produção e ação. Após a reidratação, observou-se um aumento de ROS e da autofluorescência vermelha os quais podem ser usados em futuros trabalhos na dessecação e stresse hídrico como um indicador de danos e rutura da membrana celular. Durante a desidratação, as relações hídricas das células alteram-se ocorrendo, em alguns casos, ajustamento osmótico através da acumulação de sacarose. A sacarose tem um papel importante na DT, Procedeu-se à investigação de como duas taxas de desidratação contrastantes (rápida e lenta) alteram as relações hídricas e o teor de sacarose das células de briófitos aquáticos e se essas alterações podem explicar a necessidade de desidratação lenta para induzir DT (Capítulo 5). As características das paredes celulares briófitos parecem sofrer alterações durante a desidratação rápida, permitindo à parede tornar-se mais elástica e que provavelmente permite a manutenção de um metabolismo funcional até potenciais hídricos mais reduzidos. O aumento de sacarose durante a desidratação rápida poderá ser uma tentativa de minimizar os danos quando não existe tempo para estabelecer uma proteção mais efetiva. A teoria de DT foi baseada num mecanismo de proteção constitutiva que permitiria ao briófito proteger as estruturas celulares durante a dessecação, juntamente com um mecanismo baseado em reparação após reidratação, que permitiria reparar os danos que se acumularam durante o estado de dessecação. O controlo de síntese proteica seria efetuado ao nível transcripcional através da acumulação de mRNA sendo a síntese de proteínas ativada durante a reidratação. No entanto, recentemente, o estudo de proteomas em briófitos em resposta à desidratação sugere uma nova abordagem para a DT. Procedeuse à investigação do efeito de taxas de desidratação lenta e rápida para observar os perfis proteicos, considerando ambos os processos de desidratação e reidratação (Capítulo 6). Após a desidratação rápida, os perfis de proteoma de F. antipyretica são muito semelhantes aos do controlo sem stresse. No entanto, aquando da reidratação após desidratação rápida observou-se a perda quase total das proteínas, fornecendo evidências de que o briófito não tem tempo suficiente para se preparar para a dessecação sob esta taxa de desidratação. Contudo, sob desidratação lenta ocorrem alterações substanciais nos perfis de proteoma, tanto durante a desidratação como na reidratação, o que pode indicar uma indução de mecanismos de DT nestas circunstâncias. O desenvolvimento de mecanismos de tolerância à dessecação, envolvendo a acumulação de proteínas específicas durante os ciclos de desidratação/reidratação, poderá ter permitido a colonização do meio terrestre pelos primeiros briófitos. A base para tal tolerância depende de padrões comuns de expressão de proteínas, bem como de ajustes metabólicos, os quais são muito semelhantes em briófitos de habitats distintos. Durante a desidratação, a fotossíntese é interrompida, ocorre o aumento dos níveis de açúcares solúveis, aumentam as proteínas de defesa, o citoesqueleto é desmontado e enzimas do metabolismo glicolítico aumentam. Após a reidratação, a fotossíntese reinicia, o citoesqueleto é remontado, e são mantidos elevados níveis de açúcares solúveis, enzimas do metabolismo glicolítico e proteínas de defesa. A principal conclusão sugerida por este trabalho é de que a DT ao nível celular, nomeadamente ao nível dos mecanismos moleculares, é semelhante nos diferentes briófitos independentemente do seu habitat. Além disso, estabelece que a DT é induzida por desidratação lenta sendo esta controlada até certo ponto pela morfologia do briófito, tornando-se um fator determinante na adaptação dos briófitos a cada habitat e às condições de dessecação.Fundação para a Ciência e Tecnologia (FCT, SFRH/BD/31424/2006

Using chlorophyll a fluorescence imaging to select desiccation-tolerant native moss species for water-sustainable green roofs

Green roofs have been more thoroughly investigated in the last few years due to the potential benefits they o er to ecosystems in urban areas (e.g., carbon sequestration, particle retention, heat island e ect attenuation). However, current climate change models predict an increase in desertification, with an increase in temperature and decrease in rainfall, which means there is an increasing demand for green roofs with lower water consumption. Vegetation with very little water requirements, such as desiccation-tolerant mosses, has shown a potential to complement or substitute for vascular species, increasing the sustainability of lower water use in green roofs. In this study, we use chlorophyll a fluorescence imaging to screen for bryophytes with adequate physiology to be used in green roofs placed in at-risk areas with prolonged drought episodes. Apart from Hypnum cupressiforme, all selected species presented a high potential for use in those conditions, particularly Didymodon fallax, Grimmia lisae, Pleurochaete squarrosa, and Targionia hypophylla. Chlorophyll a fluorescence imaging technology proved to be a simple and non-invasive tool for a fast screening of these poikilohydric organisms, to be used in future studies of bryophyte biology, but more importantly in the green roof industryinfo:eu-repo/semantics/publishedVersio

Acesso Aberto à literatura científica em Portugal: o passado, o presente e o futuro

Esta comunicação pretende retratar a evolução do movimento Open Access (ou “Acesso Aberto” na sua expressão portuguesa) em Portugal no decurso dos últimos dez anos, revendo o estudo “Open Access in Portugal: A State of the Art Report” (Junho de 2009), desenvolvido no âmbito do projeto Repositório Científico de Acesso Aberto de Portugal (RCAAP) e enquadrado na iniciativa Southern European Libraries Link (SELL) para aferir a situação do Acesso Aberto nos países do sul da Europa. Neste trabalho são analisados e atualizados os dados mais significativos do estudo inicial, bem como os progressos que se foram registando no que concerne à quantidade e qualidade dos repositórios estabelecidos em Portugal, a produção científica disponibilizada em Acesso Aberto nos repositórios e ainda os desenvolvimentos alcançados no domínio das revistas científicas ou académicas ativas em Portugal. A partir desta análise são ainda propostas algumas recomendações para o desenvolvimento do Acesso Aberto em Portugal e em particular na sua extensão e disseminação através de projetos colaborativos ao conjunto dos países lusófonos.This communication seeks to portray the evolution of the Open Access movement (or "Acesso Aberto" in its Portuguese terminology) in Portugal over the last ten years, reviewing the study, "Open Access in Portugal: The State of the Art Report" (June 2009), developed under the Repositório Científico de Acesso Aberto de Portugal (RCAAP) project and framed within the Southern European Libraries Link (SELL) initiative to assess the status of Open Access in southern Europe countries. We reviewed and updated the most significant data from the initial study as well as the progress registered in relation to the quantity and quality of the repositories established in Portugal, the research output available in Open Access repositories and the progress achieved in the field of academic or scientific journals active in Portugal. From this analysis we also propose some recommendations for the development of Open Access in Portugal and in particular its extension and dissemination through collaborative projects to all Portuguese speaking countries

Selecting potential moss species for green roofs in the Mediterranean Basin

Green roofs are important infrastructures to address the e ects of climate change in urban areas. However, most studies and applications have been done in cooler and wetter regions of the northern hemisphere. Climate change will lead to more extreme weather events, such as increased drought and decreased precipitation with intense flash rain events. Increase desertification is expected especially in the Mediterranean Basin, where in summer, radiation and temperature are high and water is scarce. Therefore, while vascular plants increase water consumption in green roofs during warmer periods, mosses present themselves as potential candidates due to their poikilohydric nature, responding to the environmental availability of water, completely drying out and recovering upon rehydration. Although criteria for the selection of vascular plants adapted to the Mediterranean and suitable for green roofs have been developed, no information is available regarding the selection of mosses based on scientific criteria. Here we propose selection criteria for moss species based on ecological preferences according to Ellenberg’s values and help to define moss traits suitable for a nonirrigated, nature-based green roof that tolerates the Mediterranean climate. The main result is a table of potential candidate mosses that can be either used as standalone or in conjunction with vascular plants to decrease water usage and/or manage stormwater through an easily applicable selection methodology. For green roof practitioners, we proposed that acrocarpous mosses exhibiting turf/cushion life forms and colonist or perennial life strategies best fit the requirements for such a green infrastructure in extreme climate regions with scarce water resourcesinfo:eu-repo/semantics/publishedVersio

Green roof design techniques to improve water use under Mediterranean conditions

Green roof typology can vary depending on buildings structure, climate conditions, substrate, and plants used. In regions with hot and dry summers, such as the Mediterranean region, irrigation plays an essential role, as the highest temperatures occur during the driest period of the year. Irrigation might reduce the heat island effect and improve the cooling of buildings during this period, however, the added cost of maintenance operations and additional energy consumption could outrun the benefits provided by the project. Moreover, in situations where water is scarce or primarily channelled to other uses (e.g., domestic, agriculture or industry) during drought occurrence, it is advisable to implement green roof projects with the lowest use of water possible. The objective of the present work is to investigate solutions to optimize water use in green roofs under Mediterranean conditions, such as those of southern Europe. Two case studies are presented for Portugal, and potential techniques to reduce irrigation requirements in green roofs were tested. These addressed the use of native plant species, including the extreme type of a non-irrigated green roof (Biocrust roof) and techniques for plant installation. Plant drought tolerance was found to be an advantage in green roofs under these climatic conditions and, for the species studied, aesthetic value could be maintained when irrigation decreasedinfo:eu-repo/semantics/publishedVersio

Search for top quarks accompanied by missing energy in hadronic topologies at the ATLAS experiment

Wuilleumier Pierre. Les fouilles du quartier de Choulans à Lyon. In: Bulletin de la Société Nationale des Antiquaires de France, 1948-1949, 1952. p. 186

Effects of Glyphosate-Based Herbicide on Primary Production and Physiological Fitness of the Macroalgae Ulva lactuca

The use of glyphosate-based herbicides (GBHs) worldwide has increased exponentially over the last two decades increasing the environmental risk to marine and coastal habitats. The present study investigated the effects of GBHs at environmentally relevant concentrations (0, 10, 50, 100, 250, and 500 g L1) on the physiology and biochemistry (photosynthesis, pigment, and lipid composition, antioxidative systems and energy balance) of Ulva lactuca, a cosmopolitan marine macroalgae species. Although GBHs cause deleterious effects such as the inhibition of photosynthetic activity, particularly at 250 g L1, due to the impairment of the electron transport in the chloroplasts, these changes are almost completely reverted at the highest concentration (500 g L1). This could be related to the induction of tolerance mechanisms at a certain threshold or tipping point. While no changes occurred in the energy balance, an increase in the pigment antheraxanthin is observed jointly with an increase in ascorbate peroxidase activity. These mechanisms might have contributed to protecting thylakoids against excess radiation and the increase in reactive oxygen species, associated with stress conditions, as no increase in lipid peroxidation products was observed. Furthermore, changes in the fatty acids profile, usually attributed to the induction of plant stress response mechanisms, demonstrated the high resilience of this macroalgae. Notably, the application of bio-optical tools in ecotoxicology, such as pulse amplitude modulated (PAM) fluorometry and laser-induced fluorescence (LIF), allowed separation of the control samples and those treated by GBHs in different concentrations with a high degree of accuracy, with PAM more accurate in identifying the different treatmentsinfo:eu-repo/semantics/publishedVersio

All for One: The Role of Colony Morphology in Bryophyte Desiccation Tolerance

In the last decade, several works showed that even bryophytes from aquatic environments, if slowly dehydrated, can cope with desiccation in a response like the one from desert bryophytes. This led to the hypothesis that, if bryophytes from contrasting habitats can have similar responses, desiccation tolerance (DT) is partially inductive and not only constitutive as previously proposed and, therefore, colony morphology might be the key trait responsible for controlling dehydration rate essential for DT induction. Morphology and life form may be determinant traits in the adaptation of bryophytes to habitats with different water availabilities and corresponding predicted levels in the DT inducibility spectrum. Bryophytes from habitats with different water availabilities were dried as individual shoots and as a colony. The bryophyte Fontinalis antipyretica is fully aquatic presenting a streamer life form, while the three terrestrial species present turf life form with different sizes and degrees of space between individuals in the colony. Two species were collected under trees with moist soil presenting short turf (Tortella tortuosa) and long turf (Campylopus pyriformis) life forms. Another species was completely exposed to sun light with no surrounding trees and a tall turf life form (Pleurochaete squarrosa). We used chlorophyll a fluorescence parameter Fv/Fm (maximum potential quantum efficiency of Photosystem II) as a proxy to photosynthetic fitness throughout the contrasting dehydration rates (fast and slow). These bryophytes with different life forms were submitted to an X-ray computed microtomography (µ-XCT) to assess the three-dimensional inner structure and visualize locations for water storage. Shoots dried slow or fast according to the dehydration they were exposed to, as expected, but they presented similar dehydration rates across different species. However, the aquatic moss F. antipyretica, was unable to recover from fast drying, and after 24 h the recovery following slow drying was lower than the other species. The other three species presented full recovery after 24 h, either at the individual or colony level, and either from slow or fast drying. The only exception was the colonies of Campylopus pyriformis following fast drying that presented a slightly lower recovery, probably due to a looser colony structure.info:eu-repo/semantics/publishedVersio

The use of chitosan oligosaccharide to improve artemisinin yield in well-watered and drought-stressed plants

IntroductionArtemisinin is a secondary metabolite well-known for its use in the treatment of malaria. It also displays other antimicrobial activities which further increase its interest. At present, Artemisia annua is the sole commercial source of the substance, and its production is limited, leading to a global deficit in supply. Furthermore, the cultivation of A. annua is being threatened by climate change. Specifically, drought stress is a major concern for plant development and productivity, but, on the other hand, moderate stress levels can elicit the production of secondary metabolites, with a putative synergistic interaction with elicitors such as chitosan oligosaccharides (COS). Therefore, the development of strategies to increase yield has prompted much interest. With this aim, the effects on artemisinin production under drought stress and treatment with COS, as well as physiological changes in A. annua plants are presented in this study.MethodsPlants were separated into two groups, well-watered (WW) and drought-stressed (DS) plants, and in each group, four concentrations of COS were applied (0, 50,100 and 200 mg•L-1). Afterwards, water stress was imposed by withholding irrigation for 9 days.ResultsTherefore, when A. annua was well watered, COS did not improve plant growth, and the upregulation of antioxidant enzymes hindered the production of artemisinin. On the other hand, during drought stress, COS treatment did not alleviate the decline in growth at any concentration tested. However, higher doses improved the water status since leaf water potential (YL) improved by 50.64% and relative water content (RWC) by 33.84% compared to DS plants without COS treatment. Moreover, the combination of COS and drought stress caused damage to the plant’s antioxidant enzyme defence, particularly APX and GR, and reduced the amount of phenols and flavonoids. This resulted in increased ROS production and enhanced artemisinin content by 34.40% in DS plants treated with 200 mg•L-1 COS, compared to control plants.ConclusionThese findings underscore the critical role of ROS in artemisinin biosynthesis and suggest that COS treatment may boost artemisinin yield in crop production, even under drought conditions