Sands through the hourglass the structural and functional diversity of major intrinsic proteins

Major Intrinsic Proteins (MIPs) are an ancient family of integral membrane proteins that mediate the bidirectional flux of water and small solutes across cellular membranes. Genomic and phylogenetic analyses indicate that plants contain more MIP genes than their animal and microbial counterparts. An analysis of MIP structure also indicates that plant MIPs structurally diverse at the regions that control selectivity of these proteins. Homology modeling was performed using all 35 members of the MIP family from Arabidopsis thaliana. This analysis revealed that MIPs can be divided into 8 functional subgroups based on the amino acids in their selectivity determining ar/R regions. A broader phylogenetic analysis of all available MIP sequences indicates that 92 ar/R regions exist in this dataset, and that much of the diversity arises from plant sources.Homology modeling indicated that the Nodulin 26-like intrinsic protein (NIP) family of Arabidopsis could be divided into two subgroups based on ar/R classification: NIP subgroup I and II. Functional analysis indicates that these two subgroups are functionally distinct. NIP subgroup I forms aquaglyceroporin channels that are also permeable to ammonia, while NIP subgroup II channels are impermeable to water and capable of transporting larger solutes, such as urea. Site-directed mutagenesis studies were used to rationally interconvert the selectivity of these proteins by amino acid substitutions in the ar/R region. Finally, it was demonstrated that members of NIP subgroup II in Arabidopsis form physiologically relevant boric acid channels at the plasma membrane. Nodulin 26, the archetypal NIP, is phosphorylated by a calcium dependant protein kinase (CDPK) at Ser 262 in its C-terminus. This study demonstrates that nodulin 26 phosphorylation increases the water permeability of the channel.In addition, the C-terminus was found to constitute a protein interaction site for nodule cytosolic glutamine synthetase. Finally, the study was extended to characterize MIP channels from the common pea aphid (ApAQP1 and ApAQP2). The results indicate that ApAQP1 is a water-selective aquaporin that is involved in aphid gut osmoregulation, while ApAQP2 is a water channel that is permeable to an array of linear polyols. The potential physiological function of this channel is also discussed

Soybean Nodulin 26: A Channel for Water and Ammonia at the Symbiotic Interface of Legumes and Nitrogen-fixing Rhizobia Bacteria

During the infection and nodulation of legume roots by soil bacteria of the Rhizobiaceae family, the invading endosymbiont becomes enclosed within a specialized nitrogen-fixing organelle known as the symbiosome . In mature nodules the host infected cells are occupied by thousands of symbiosomes, which constitute the major organelle within this specialized cell type. The symbiosome membrane is the outer boundary of this organelle which controls the transport of metabolites between the symbiont and the plant host. These transport activities include the efflux of the primary metabolic product of nitrogen fixation and the uptake of dicarboxylates as an energy source to support bacterial nitrogen fixation. Soybean nodulin 26, a member of the aquaporin superfamily, is the major protein component of the symbiosome membrane that encloses nitrogen-fixing bacteroids in root nodules. Previous work has demonstrated that nodulin 26 facilitates the transport of water as well as other uncharged solutes such as glycerol and formamide. In addition, it is clear that the protein is a target for developmental and environmental sensitive posttranslational phosphorylation which may regulate transport activity. The present research project provides evidence that nodulin 26 is an “aquaglycero-ammoniaporin” that is specifically localized to the symbiosome membrane, where it could play a potential role in osmoregulatory and metabolic functions in the symbiosis. First, it is shown that purified nodulin 26 reconstituted into liposomes possesses an ammonia permease activity that is favored approximately 4-fold over its aquaporin activity. Second, it is shown that nodulin 26 serves as a docking site for the ammonia assimilatory enzyme, cytosolic glutamine synthetase on the surface of the symbiosome membrane. Third, it is shown that phosphorylation of nodulin 26 exerts opposite effects on the regulation of ammonia and water transport activities. Fourth, it is demonstrated that phosphorylation of nodulin 26 in mature nitrogen-fixing nodules is tightly controlled by various environmental osmotic stimuli that regulate the rate of nitrogen-fixation as well as modulation of the oxygen diffusion barrier inside nodules. A model for how nodulin 26 phosphorylation could contribute to the regulation of these physiological processes is devised

Two L-histidine transporters and one sensor kinase

The Gram-negative soil bacterium Pseudomonas putida KT2440 colonizes the rhizosphere of plants and promotes their growth. It is genetically accessible, solvent-resistant and biosafe and therefore, serves as a platform for industrial biosyntheses (for example, plastic-like polymers and biosurfactants). The bacterium is metabolically extremely versatile and can utilize diverse nutrients as carbon and nitrogen sources. Two-component systems consisting of a sensor kinase and a response regulator play an important role in the adaptation of the cell metabolism to environmental and nutrient conditions. These regulatory systems control, among other things, the expression of genes encoding nutrient-specific transporters. The focus of this thesis is on the two-component system CbrA/CbrB and the transporter HutT of P. putida KT2440. CbrA/CbrB is a key regulatory system involved in catabolite repression and control of various catabolic pathways in other pseudomonads. Remarkably, in addition to sensor kinase-typical domains, the sensor kinase CbrA also contains a membrane-integral transporter domain that belongs to the solute:Na+ symporter family (SSSF). The physiological significance and molecular function of the SSSF domain of CbrA are unknown. HutT is also a protein localized in the cytoplasmic membrane and belongs to the amino acid transporter (AAT) family within the amino acid polyamine organocation (APC) transporter superfamily. The function of HutT in P. putida KT2440 has not been studied experimentally before. The task of this thesis was to elucidate the functional properties of CbrA/CbrB and HutT of P. putida KT2440. This investigation confirmed also for P. putida KT2440 that the CbrA/CbrB system is involved in catabolite repression by regulating the expression of the small RNA CrcZ. Furthermore, the two-component system controls the catabolism of basic amino acids such as histidine. For biochemical analyses, CbrA and CbrB of P. putida KT2440 were produced heterologously in Escherichia coli and purified. Experiments with cells and purified proteins revealed that CbrA can bind and transport histidine. The transport was found to be specific for histidine. The inhibition pattern obtained with ionophores suggests an electrochemical proton gradient as the driving force. The kinase domain was not required for the uptake of histidine by the SSSF domain of CbrA and did not significantly affect transport kinetics. In addition, the histidine kinase domain was experimentally shown to autophosphorylate and transfer the phosphoryl group to the response regulator CbrB. The SSSF domain was not essential for these activities but modulated autokinase activity. Phosphatase activity of CbrA was not detectable. None of the phosphotransfer activities are significantly affected by L-histidine. The results indicate that CbrA functions as an L-histidine transporter and sensor kinase in P. putida KT2440. For HutT, this thesis showed that deletion of the corresponding gene inhibited the growth of P. putida KT2440 on histidine. This suggests that the transporter plays a central role in the uptake of histidine in this bacterium. Transport experiments with cells and purified and reconstituted protein revealed that HutT is a histidine:H+ symporter. Analyses of histidine binding and transport kinetics, as well as competition experiments, showed that HutT is highly specific for histidine and binds it with high affinity. Substitutional analyses provided evidence for amino acids possibly involved in substrate binding or coupling of histidine transport to an electrochemical proton gradient. In summary, the sensor kinase CbrA plays a central role as a metabolic regulator and transports the amino acid histidine in P. putida KT2440. The physiological significance of this transport remains unclear. HutT, on the other hand, plays a central role in the uptake of histidine when the amino acid is used as a carbon source.Das Gram-negative Bodenbakterium Pseudomonas putida KT2440 besiedelt die Rhizosphäre von Pflanzen und fördert deren Wachstum. Es ist genetisch zugänglich, lösungsmittelbeständig, und biologisch sicher und dient deshalb als Plattform für industrielle Biosynthesen (zum Beispiel kunststoffähnliche Polymere und Biotenside). Das Bakterium ist metabolisch äusserst versatil und kann vielfältige Nährstoffe als Kohlenstoff- und Stickstoffquelle nutzen. Bei der Anpassung des Stoffwechsels an Umwelt- und Nährstoffbedingungen spielen Zwei-Komponenten-Systeme bestehend aus einer Sensorkinase und einem Antwortregulator eine wichtige Rolle. Diese Regulationssysteme kontrollieren unter anderem die Expression von Genen, die für nährstoffspezifische Transporter kodieren. Im Mittelpunkunkt dieser Arbeit stehen das Zweitkomponenten-System CbrA/CbrB und der Transporter HutT von P. putida KT2440. CbrA/CbrB ist ein zentrales regulatorisches System, das in anderen Pseudomonaden unter anderem an der Katabolitrepression und an der Kontrolle verschiedener kataboler Stoffwechselwege beteiligt ist. Bemerkenswert ist, dass die Sensorkinase CbrA neben Sensorkinase-typischen Domänen auch eine membranintegrale Transporterdomäne enthält, die zur Substrat:Na+-Symporter-Familie (SSSF) gehört. Physiologische Bedeutung und molekulare Funktion der SSSF-Domäne von CbrA sind nicht bekannt. HutT ist ebenfalls ein in der Zytoplasmamembran lokalisiertes Protein, das innerhalb der Superfamilie der Aminosäure-Polyamin-Organokationen (APC) Transporter zur Aminosäuretransporter (AAT)-Familie gehört. Die Funktion von HutT in P. putida KT2440 wurde bisher noch nicht experimentell untersucht. Aufgabe der Arbeit war die Aufklärung der funktionellen Eigenschaften von CbrA/CbrB und HutT von P. putida KT2440. In dieser Arbeit wurde auch für P. putida KT2440 nachgewiesen, dass das CbrA/CbrB-System an der Katabolitrepression durch Regulierung der Expression der kleinen RNA CrcZ beteiligt ist. Weiterhin kontrolliert das Zweikomponenten-System den Katabolismus von basischen Aminosäuren wie zum Beispiel L-Histidin. Für biochemische Analysen wurden CbrA und CbrB von P. putida KT2440 heterolog in Escherichia coli produziert und gereinigt. Untersuchungen mit Zellen und gereinigten Proteinen zeigten, dass CbrA L-Histidin binden und transportieren kann. Der Transport erwies sich als spezifisch für L-Histidin. Das mit Ionophoren erhaltene Inhibitionsmuster wies auf einen elektrochemischen Protonengradienten als Triebkraft hin. Die Kinasedomäne war für die Aufnahme von L-Histidin durch die SSSF-Domäne von CbrA nicht erforderlich und hatte keinen wesentlichen Einfluss auf die Transportkinetik. Darüber hinaus wurde experimentell nachgewiesen, dass sich die Sensorkinase autophosphoryliert und die Phosphorylgruppe auf den Antwortregulator CbrB überträgt. Die SSSF-Domäne war für diese Aktivitäten nicht essentiell, modulierte aber die Autokinase-Aktivität. Eine Phosphataseaktivität von CbrA war nicht nachweisbar. Keine der Phosphotransferaktivitäten wird durch L-Histidin signifikant beeinflusst. Die Ergebnisse zeigen, dass CbrA als L-Histidin-Transporter und Sensorkinase in P. putida KT2440 fungiert. Für HutT wurde in dieser Arbeit gezeigt, dass die Deletion des entsprechenden Gens das Wachstum von P. putida KT2440 auf Histidin inhibiert. Dies deutet darauf hin, dass der Transporter eine zentrale Rolle bei der Aufnahme von Histidin in diesem Bakterium spielt. Transportexperimente mit Zellen und gereinigtem und rekonstituierten Protein ergaben, dass HutT ein Histidin:H+ -Symporter ist. Analysen von Histidinbindung und Transportkinetik sowie Kompetitionsexperimente zeigten, dass HutT hochspezifisch für Histidin ist und dieses mit hoher Affinität bindet. Durch Substitutionsanalysen wurden Hinweise auf Aminosäuren erhalten, die möglicherweise an der Substratbindung oder der Kopplung an einen elektrochemischen Protonengradienten beteiligt sind. Zusammenfassend ist festzustellen, dass die Sensorkinase CbrA in P. putida KT2440 eine zentrale Rolle als Stoffwechselregulator spielt und die Aminosäure Histidin transportiert. Die physiologische Bedeutung dieses Transports bleibt unklar. HutT hingegen spielt eine zentrale Rolle bei der Aufnahme von Histidin, wenn die Aminosäure als Kohlenstoffquelle genutzt wird

The role of vitamin D and β-defensin 103 on skin: from defence to pigmentation regulation

294 p.El objetivo principal de esta tesis era investigar la posible relación entre la pigmentación de la piel y el sistema inmunitario en humanos. Para ello, hemos analizado por un lado la diversidad del gen DEFB103 que codifica un péptido antimicrobiano, la expresión de este gen por los queratinocitos, y la influencia del péptido sobre los melanocitos. Por otro lado, hemos analizado el efecto de la vitamina D sobre el los melanocitos, mediante RNA-Seq. Con este trabajo demostramos que la pigmentación de la piel puede ser modulada por moléculas que están implicadas en el sistema inmunitario. En particular la beta-defensinas 103 y la vitamina D activa, las cuales hemos visto que tienen la capacidad de influir en la expresión de genes melanogénicos. Además, el efecto de ambos factores parece estar relacionado con los niveles de pigmentación. El efecto de la vitamina D en los melanocitos es diferente con respecto al fenotipo pigmentario y su efecto en la pigmentación parece ser solo en melanocitos ligeramente pigmentadosThis work was supported by a predoctoral fellowship from the Basque Government to Arrate Sevilla (PRE_2014_1_419), an EMBO Short-Term Fellowship (7014), a MINECO proyect grant (CGL2014-58526-P), by FEDER (Fondo Europeo de Desarrollo Regional), and by projects from the Basque Goverment (IT1138-16 and SAIOTEK2012: S-PE12UN051)

The role of vitamin D and β-defensin 103 on skin: from defence to pigmentation regulation

294 p.El objetivo principal de esta tesis era investigar la posible relación entre la pigmentación de la piel y el sistema inmunitario en humanos. Para ello, hemos analizado por un lado la diversidad del gen DEFB103 que codifica un péptido antimicrobiano, la expresión de este gen por los queratinocitos, y la influencia del péptido sobre los melanocitos. Por otro lado, hemos analizado el efecto de la vitamina D sobre el los melanocitos, mediante RNA-Seq. Con este trabajo demostramos que la pigmentación de la piel puede ser modulada por moléculas que están implicadas en el sistema inmunitario. En particular la beta-defensinas 103 y la vitamina D activa, las cuales hemos visto que tienen la capacidad de influir en la expresión de genes melanogénicos. Además, el efecto de ambos factores parece estar relacionado con los niveles de pigmentación. El efecto de la vitamina D en los melanocitos es diferente con respecto al fenotipo pigmentario y su efecto en la pigmentación parece ser solo en melanocitos ligeramente pigmentadosThis work was supported by a predoctoral fellowship from the Basque Government to Arrate Sevilla (PRE_2014_1_419), an EMBO Short-Term Fellowship (7014), a MINECO proyect grant (CGL2014-58526-P), by FEDER (Fondo Europeo de Desarrollo Regional), and by projects from the Basque Goverment (IT1138-16 and SAIOTEK2012: S-PE12UN051)

Dynamics of protein-drug interactions inferred from structural ensembles and physics-based models

The conformational flexibility of target proteins is a major challenge in understanding and modeling protein-drug interactions. A fundamental issue, yet to be clarified, is whether the observed conformational changes are controlled by the protein, or induced by the inhibitor. While the concept of induced fit has been widely adopted for describing the structural changes that accompany ligand binding, there is growing evidence in support of the dominance of proteins' intrinsic dynamics, which has been evolutionarily optimized to accommodate its functional interactions. The wealth of structural data for target proteins in the presence of different ligands now permits us to make a critical assessment of the balance between these two effects in selecting the bound forms. We focused on three widely studied drug targets, HIV-1 reverse transcriptase, p38 MAP kinase, and cyclin-dependent kinase 2. A total of 292 structures determined for these enzymes in the presence of different inhibitors as well as unbound form permitted us to perform an extensive comparative analysis of the conformational space accessed upon ligand binding, and its relation to the intrinsic dynamics prior to ligand binding as predicted by elastic network model analysis. Further, we analyzed NMR ensembles of ubiquitin and calmodulin representing their microseconds range solution dynamics. Our results show that the ligand selects the conformer that best matches its structural and dynamic properties amongst the conformers intrinsically accessible to the protein in the unliganded form. The results suggest that simple but robust rules encoded in the protein structure play a dominant role in pre-defining the mechanisms of ligand binding, which may be advantageously exploited in designing inhibitors. We apply these lessons to the study of MAP kinase phosphatases (MKPs), which are therapeutically relevant but challenging signaling enzymes. Our study provides insights into the interactions and selectivity of MKP inhibitors and shows how an allosteric inhibition mechanism holds for a recently discovered inhibitor of MKP-3. We also provide evidence for the functional significance of the structure-encoded dynamics of rhodopsin and nicotinic acetylcholine receptor, members of two membrane proteins classes serving as targets for more than 40% of all current FDA approved drugs

RNA-SEQ applied to the peacock blenny Salaria pavo: unveiling the gene networks and signalling pathways behind phenotypic plasticity in a littoral fish

Phenotypic plasticity is the ability of an individual genome to produce different phenotypes depending on environmental cues. These plastic responses rely on diverse genomic mechanisms and allow an organism to maximize its fitness in a variety of social and physical settings. The development of next-generation sequencing (NGS) technologies, especially RNA Sequencing (RNA-Seq), has made it possible to investigate the distinct patterns of gene expression known to be underlying plastic phenotypes in species with ecological interest. In teleost fishes, changes in phenotypes is often observed during the reproductive season, with shifts and adjustments in dominance status that can lead to the co-existence of multiple reproductive morphs within the same population. One such example is the peacock blenny Salaria pavo (Risso, 1810), a species where the intensity of mating competition varies among populations due to nest-site availability, such that two different levels of plasticity arise: 1) intraspecific variation in reproductive behaviour for males that can follow either of two developmental pathways, grow directly into nest-holder males, or behave first as female mimics to sneak fertilizations (sneaker males) and later transition into nest-holder males, and 2) inter-population variation in courting roles of females and nest-holder males. This system provides the ideal basis to apply RNA-Seq methods to study plasticity since differences in reproductive traits within and among populations can reveal which genetic and genomic mechanisms underpin the observed variation in behavioural response to changes in the social environment. However, the genomic information available for this species was scarce, and hence multiple sequencing techniques were used and the methodologies applied optimized throughout the work. In this thesis, we start by first obtaining a de novo transcriptome assembly to develop the first genetic markers for this species (Chapter 2). These microsatellites were used to elucidate the reproductive success (i.e. consisting of mating success and fertilization success) of male ARTs, which can be used as a proxy of Darwinian fitness (Chapter 3). In this study, we detected a fertilization success for nestholder males of 95%, and showed a stronger influence of the social environment rather than morphological variables in the proportion of lost fertilizations by nest-holder males of this species. Taking advantage of the developed transcriptome, we used highthroughput sequencing to obtain expression profiles for male morphs (i.e. intraspecific variation) and females in this species, and focus on the role of differential gene expression in the evolution of sequential alternative reproductive tactics (ARTs) that involve the expression of both male and female traits (Chapter 4). Additionally, we show how the distinct behavioural repertoires are facilitated by distinct neurogenomic states, which discriminate not only sex but also male morphs. Lastly, using two different target tissues, gonads and forebrain, we focus on the genomic regulation of sex roles in courtship behaviour between females and males from two populations under different selective regimes (inter-population variation), the Portuguese coastal population with reversed sex roles and the rocky Italian population with ‘conventional’ sex roles (Chapter 5). Here we demonstrate that variation in gene expression at the brain level segregates individuals by population rather than by sex, indicating that plasticity in behaviour across populations drives variation in neurogenomic expression. On the other hand, at the gonad level, variation in gene expression segregates individuals by sex and then by population, indicating that sexual selection is also acting at the intrasexual level, particularly in nestholder males by paralleling differences in gonadal investment. However, the genomic mechanisms underlying courtship behaviour were not fully elucidated, and more studies are necessary.A plasticidade fenotípica consiste na capacidade de o mesmo genoma produzir diferentes fenótipos comportamentais dependendo das pistas ambientais recebidas. Estas respostas plásticas dependem de diversos mecanismos genómicos e permitem que o indivíduo maximize a sua fitness (aptidão) numa variedade de ambientes ecológicos. O desenvolvimento verificado nas tecnologias de sequenciação de alto desempenho ao longo da última década, globalmente denominadas de “Next Generation Sequencing” (NGS), permitiu o estabelecimento de métodos de análise e ferramentas genómicas que podem ser aplicadas em todos os sistemas ecológicos de interesse em biologia, sem a existência prévia de um genoma curado. Nomeadamente a tecnologia de sequenciação de ARN, conhecida globalmente como RNA-Seq, tornou possível a investigação dos perfis de expressão génica que se sabe serem determinantes na emergência de fenótipos plásticos, e consequentemente permitem determinar fenótipos em estados distintos de expressão genómica. Em peixes teleósteos, é possível observar com frequência modificações no fenótipo comportamental durante o período de reprodução, como por exemplo alterações e ajustes no estatuto de dominância que podem levar à coexistência de indivíduos que apresentam diferentes táticas de reprodução dentro da mesma população. Um desses exemplos é o peixe marachomba-pavão Salaria pavo (Risso, 1810), onde a intensidade na competição intra e intersexual varia entre populações sendo modulada pela disponibilidade de locais de nidificação, de forma a que dois níveis diferentes de plasticidade surgem: 1) variação intraespecífica no comportamento reprodutivo em machos que podem seguir uma de duas vias de desenvolvimento, investirem no seu crescimento e tornarem-se machos nidificantes na sua primeira época de reprodução, ou primeiro seguir uma tática de macho parasita onde investem em fertilizações furtivas, sendo que mais tarde no seu desenvolvimento fazem a transição para macho nidificante, e 2) variação interpopulacional nos papeis de corte de fêmeas e machos nidificantes. Os machos parasitas, conhecidos nesta espécie como “sneakers”, possuem uma particularidade que os tornam singulares, para além de imitarem a morfologia das fêmeas também conseguem imitar o seu comportamento de corte direcionado ao macho nidificante, o que lhes permite aproximarem-se discretamente dos ninhos dos machos e fertilizar parte dos ovos que as fêmeas depositam. Este sistema constitui a base ideal para aplicar métodos de RNA-Seq e estudar esta plasticidade fenotípica, uma vez que diferenças nas características reprodutivas dentro e entre populações podem revelar quais os mecanismos genéticos e genómicos subjacentes à variação observada em resposta a mudanças no ambiente ecológico. No entanto, a informação genómica disponível nesta espécie é reduzida e, por isso diferentes técnicas de sequenciação, assim como diferentes métodos de análise foram usados e otimizados ao longo deste trabalho. A presente tese é constituída por quatro trabalhos, sendo que no primeiro estudo se começa pela sequenciação de uma biblioteca de ARN proveniente de uma mistura de múltiplos indivíduos e de tecidos, de forma a captar a diversidade genética e desenvolver os primeiros marcadores genéticos nesta espécie (Capítulo 2). Com base nestes marcadores, microssatélites polimórficos, foi possível genotipar uma fração dos indivíduos da população existente na Ilha da Culatra (Ria Formosa, Portugal) bem como os ovos retirados de ninhos alvo, de forma a fazer análises de paternidade (Capítulo 3). Neste estudo, foi possível estimar o sucesso de fertilização de ovos de cada uma das táticas alternativas de reprodução, e usá-la como medida representativa de fitness de cada tática alternativa de reprodução nesta espécie. Os resultados indicam um sucesso de fertilização para os machos nidificantes de 95%, e mostramos que existe uma maior influência do ambiente social do que de variáveis morfológicas na proporção de fertilizações não obtidas pelos machos nidificantes, quando comparado com estudos anteriores. Usando o transcriptoma obtido no primeiro trabalho, avançámos com a caraterização genómica de cada um dos fenótipos presentas na população da ilha da Culatra, fêmeas, machos nidificantes, machos sneakers e machos de transição (machos que apenas investem no seu crescimento, não se reproduzindo, e consequente transição de sneaker para macho nidificante) (Capítulo 4). Para tal, foi sequenciado em profundidade o transcriptoma de cérebro de cada um deste fenótipos, e os perfis de expressão obtidos para machos e fêmeas desta espécie, onde o foco do estudo se centrava no papel da expressão génica diferencial na evolução de táticas reprodutivas alternativas sequenciais que envolvem a expressão de ambos os traços masculinos e femininos. Os resultados obtidos, mostram como repertórios comportamentais distintos são facilitados por estados neurogenómicos distintos, que discriminam não apenas o sexo, mas também as táticas alternativas de reprodução. Por fim, utilizando dois tecidos-alvo, gónadas e prosencéfalo, focámo-nos na regulação genómica dos papeis sexuais no comportamento de corte entre fêmeas e machos nidificantes de duas populações sob diferentes regimes seletivos, a população costeira portuguesa com papeis sexuais invertidos e a população rochosa italiana, com papeis sexuais ‘convencionais’ (Capítulo 5). Os resultados obtidos mostram que ao nível do cérebro, a variação na expressão génica segrega os indivíduos por população e não por sexo, indicando que a plasticidade no comportamento entre as populações induz uma maior variação na expressão neurogenómica. Por outro lado, ao nível das gónadas, a variação na expressão génica segrega os indivíduos por sexo e também por população, indicando que a seleção sexual está a atuar ao nível intrasexual, particularmente nos machos nidificantes, indo de encontro a diferenças detetadas entre populações no investimento alocado às gónadas. No entanto, os mecanismos genómicos subjacentes ao comportamento de corte não foram totalmente elucidados, e mais estudos são necessários.The work presented here was developed at Instituto Gulbenkian de Ciência (IGC) in Oeiras, with the support of both ISPA – Instituto Universitário in Lisbon, for the maintenance of live fish, and Centro de Ciências do Mar (CCMAR) at Universidade do Algarve, for logistics and support during fieldwork in Ria Formosa

Morphological, physiological, and transcriptional responses of Populus trichocarpa to water deficit stress

Under the threat of climate change with continued fossil fuel use, investment in developing renewable energies is increasingly important. One category of renewable energy sources, biofuels, is well suited to industries that require a liquid fuel source. However, producing biofuel feedstocks is challenging from a sustainability perspective, as they require extensive land area and resource input to grow on marginal lands. The identification of strategies that feedstock species use to maintain productivity with limited to no resource inputs could aid in the selection of optimal genotypes or targets of breeding or engineering efforts to maximize productivity in feedstock plantations. Some of these targets may be belowground traits, which have not been extensively studied. Therefore, this research sought to uncover and compare the aboveground and belowground strategies used by two Populus trichocarpa genotypes differing in their tolerance to water deficit stress in response to drying. Trees were subjected to gradual drying to wilt point followed by three weeks of consistent stress. Aboveand belowground morphology and physiology were measured at wilt point and at the end of the experiment, and the leaf and root transcriptomes were sequenced to provide insight into the metabolic mechanisms underlying responses to water deficit stress. The tolerant genotype kept its stomata partially open under stress, allowing more water loss via transpiration but also enabling carbon uptake. It compensated for continued water loss by devoting resources to root growth for soil exploration for water, particularly using finer roots. The balance of resource use and acquisition likely allowed for better maintenance of productivity during stress. At the transcriptional level, jasmonate signaling pathways, which cause similar morphological and physiological changes in other species, were upregulated in the tolerant genotype. Results of this study suggest that root characteristics that promote water uptake from a limited pool of resources can allow for continued gas exchange under stress, which has been previously identified as a sign of sensitivity, to promote drought tolerance. Additionally, a need to investigate the role of jasmonate signaling in drought tolerance in poplar as a potential target of feedstock improvement is revealed