Is geographical variation driving the transcriptomic responses to multiple stressors in the kelp Saccharina latissima

Background: Kelps (Laminariales, Phaeophyceae) are brown macroalgae of utmost ecological, and increasingly economic, importance on temperate to polar rocky shores. Omics approaches in brown algae are still scarce and knowledge of their acclimation mechanisms to the changing conditions experienced in coastal environments can benefit from the application of RNA-sequencing. Despite evidence of ecotypic differentiation, transcriptomic responses from distinct geographical locations have, to our knowledge, never been studied in the sugar kelp Saccharina latissima so far. Results: In this study we investigated gene expression responses using RNA-sequencing of S. latissima from environments with contrasting temperature and salinity conditions – Roscoff, in temperate eastern Atlantic, and Spitsbergen in the Arctic. Juvenile sporophytes derived from uniparental stock cultures from both locations were pre-cultivated at 8 °C and SA 30. Sporophytes acclimated to 0 °C, 8 °C and 15 °C were exposed to a low salinity treatment (SA 20) for 24 h. Hyposalinity had a greater impact at the transcriptomic level than the temperature alone, and its effects were modulated by temperature. Namely, photosynthesis and pigment synthesis were extensively repressed by low salinity at low temperatures. Although some responses were shared among sporophytes from the different sites, marked differences were revealed by principal component analysis, differential expression and GO enrichment. The interaction between low temperature and low salinity drove the largest changes in gene expression in sporophytes from Roscoff while specimens from Spitsbergen required more metabolic adjustment at higher temperatures. Moreover, genes related to cell wall adjustment were differentially expressed between Spitsbergen and Roscoff control samples. Conclusions: Our study reveals interactive effects of temperature and salinity on transcriptomic profiles in S. latissima. Moreover, our data suggest that under identical culture conditions sporophytes from different locations diverge in their transcriptomic responses. This is probably connected to variations in temperature and salinity in their respective environment of origin. The current transcriptomic results support the plastic response pattern in sugar kelp which is a species with several reported ecotypes. Our data provide the baseline for a better understanding of the underlying processes of physiological plasticity and may help in the future to identify strains adapted to specific environments and its genetic control

Genetics and Genomics Studies for Water-related Stresses in Rice (Oryza sativa L.)

Rice is one of the most important crops that feeds more than half of the world’s population. Along with the escalating problems with climate change, drought and submergence events have increased in recent years and have challenged the sustainability of rice production. This dissertation focused on these two water-related stresses, and studied the tolerance mechanisms underlying. Reproductive stage is the most water-sensitive period for rice cultivation, therefore the study was conducted to investigate the molecular and physiological responses of two rice genotypes, ‘Rondo’ and ‘4610’, to drought stress during reproductive stage under field conditions. Leaf samples were collected for RNA-Seq. Additionally, 10 agronomic traits and chlorophyll fluorescence were measured. The results showed that 4610 had better performance than Rondo under moderate drought stress conditions. The results indicated that 4610 had more stress responsive GO terms and several known genes families related to drought stress were also identified in 4610 up-regulated DEGs, including the LEA proteins, HSPs, APXs, and GSTs. The second goal was to characterize the region of qSub8.1, a new submergence tolerance QTL during vegetative stage, which will enable us to develop suitable DNA markers and identify the gene(s) underlying qSub8.1 for further functional characterization. Three rice varieties, Ciherang-Sub1, Ciherang, and IR64-Sub1, were sequenced with 150 bp pair-end WGS. The results showed that Ciherang-Sub1 genome is composed of 59% Ciherang, 24% of IR64-Sub1, and 17% of unknown sources; and the qSub8 region is mainly from Ciherang with a few introgressed segments from IR64- Sub1 and unknown sources. There are challenges in conducting plant transformation and regeneration required to introduce the CRISPR reagents into the plant cell for gene-editing. Here, we used mature seeds as explants and reported a high-efficiency transformation and regeneration protocol for a recalcitrant indica rice cultivar Ciherang-Sub1 using particle bombardment to deliver CRISPR/Cas9 gene-editing vector. Our protocol successfully gives an optimal condition for shoot regeneration with 95% of regeneration rate and by using this protocol, CRISPR gene-edited plants can be generated and validated within approximately 12 weeks. Using this protocol, CRISPR gene-edited plants can be generated and validated within 12 weeks

Interactive effects of iron and light limitation on the molecular physiology of the Southern Ocean diatom Fragilariopsis kerguelensis

The polar diatom Fragilariopsis kerguelensis is ubiquitous in Southern Ocean waters and is a major responder to iron fertilization, encountering large gradients in iron concentrations and light availability. We performed a comparative transcriptomic analysis of F. kerguelensis grown under varying iron and light conditions in order to investigate the molecular underpinnings that may explain its physiological response to iron and light limitation. Low iron reduced growth rates more than low light, but there was not an additive effect of low iron and low light on growth rate. Low iron treatments (saturating and low light) had the largest transcriptomic response; however, expression patterns were more similar in low light treatments (high and low iron). Under iron and light limitation, carbon fixation and amino acid, ribosome, and sulfur metabolisms were overrepresented relative to the control (iron replete, saturating light). Transcripts of genes encoding for the proteins aquaporin, proteorhodopsin, plastocyanin, and flavodoxin were overrepresented to the greatest extent in the low iron/low light treatment, indicating there may be an additive effect of iron/light colimitation at the molecular level. Iron and light replete cells demonstrated increased expression of genes encoding for the proteins ferritin, carbonic anhydrase, and numerous iron-dependent proteins relative to the growth-limiting treatments. This transcriptome analysis reveals mechanisms that may underpin the ecological success of this diatom in low iron and light environments, highlighting the important role of diversified photosynthetic isoforms, iron acquisition, unique detoxification mechanisms of reactive oxygen species, and metabolic shifts in amino acid recycling and carbon metabolism

Synthetic biology approaches to engineer saccharomyces cerevisiae towards the industrial production of valuable polyphenolic compounds

Polyphenols are plant secondary metabolites with diverse biological and potential therapeutic activities such as antioxidant, anti-inflammatory and anticancer, among others. However, their extraction from the native plants is not enough to satisfy the increasing demand for this type of compounds. The development of microbial cell factories to effectively produce polyphenols may represent the most attractive solution to overcome this limitation and produce high amounts of these bioactive molecules. With the advances in the synthetic biology field, the development of efficient microbial cell factories has become easier, largely due to the development of the molecular biology techniques and by the identification of novel isoenzymes in plants or simpler organisms to construct the heterologous pathways. Furthermore, efforts have been made to make the process more profitable through improvements in the host chassis. In this review, advances in the production of polyphenols by genetically engineered Saccharomyces cerevisiae as well as by synthetic biology and metabolic engineering approaches to improve the production of these compounds at industrial settings are discussed.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 – North Portugal Regional Program. In addition, this research has been carried out at the Biomass and Bioenergy Research Infrastructure (BBRI)–LISBOA-010145-FEDER-022059, supported by Operational Program for Competitiveness and Internationalization (PORTUGAL2020), the Lisbon Portugal Regional Operational Program (Lisboa2020), and Norte2020 under the Portugal 2020 Partnership Agreement, through the ERDF. JR is recipient of a fellowship supported by a doctoral advanced training (SFRH/BD/138325/2018) funded by FCT.info:eu-repo/semantics/publishedVersio

Unraveling the Physiological Roles of the Cyanobacterium Geitlerinema sp. BBD and Other Black Band Disease Community Members through Genomic Analysis of a Mixed Culture

Black band disease (BBD) is a cyanobacterial-dominated polymicrobial mat that propagates on and migrates across coral surfaces, necrotizing coral tissue. Culture-based laboratory studies have investigated cyanobacteria and heterotrophic bacteria isolated from BBD, but the metabolic potential of various BBD microbial community members and interactions between them remain poorly understood. Here we report genomic insights into the physiological and metabolic potential of the BBD-associated cyanobacterium Geitlerinema sp. BBD 1991 and six associated bacteria that were also present in the non-axenic culture. The essentially complete genome of Geitlerinema sp. BBD 1991 contains a sulfide quinone oxidoreductase gene for oxidation of sulfide, suggesting a mechanism for tolerating the sulfidic conditions of BBD mats. Although the operon for biosynthesis of the cyanotoxin microcystin was surprisingly absent, potential relics were identified. Genomic evidence for mixed-acid fermentation indicates a strategy for energy metabolism under the anaerobic conditions present in BBD during darkness. Fermentation products may supply carbon to BBD heterotrophic bacteria. Among the six associated bacteria in the culture, two are closely related to organisms found in culture-independent studies of diseased corals. Their metabolic pathways for carbon and sulfur cycling, energy metabolism, and mechanisms for resisting coral defenses suggest adaptations to the coral surface environment and biogeochemical roles within the BBD mat. Polysulfide reductases were identified in a Flammeovirgaceae genome (Bacteroidetes) and the sox pathway for sulfur oxidation was found in the genome of a Rhodospirillales bacterium (Alphaproteobacteria), revealing mechanisms for sulfur cycling, which influences virulence of BBD. Each genomic bin possessed a pathway for conserving energy from glycerol degradation, reflecting adaptations to the glycerol-rich coral environment. The presence of genes for detoxification of reactive oxygen species and resistance to antibiotics suggest mechanisms for combating coral defense strategies. This study builds upon previous research on BBD and provides new insights into BBD disease etiology

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Nanopartículas de óxido de zinco sub-tóxicas modulam os níveis das proteínas PSD-95 e shank 3

The effect of zinc on neuronal systems is growing interest in scientific research due to a possible role as a modulator of synaptic activity. Additionally, recent research points to the therapeutic potential of zinc oxide nanoparticles (ZnO NPs) on neuronal cells. These nanoparticles are excellent drug carriers to the brain because, besides having positive characteristics for the protection of cellular homeostasis, they are small, allowing them to pass the blood brain barrier and interact directly within these cells. In this sense, the present thesis explored the effect of sub-toxic ZnO NP concentrations on cell viability and ROS production in SH-SY5Y cells, as well as their impact on the expression levels of PSD-95, SHANK 3 and β-actin proteins. The results showed increases in PSD-95 and SHANK 3 protein expression without variations in β-actin expression after neuronal cell exposure to sub-toxic concentrations and at reduced exposure times to ZnO NPs. Future efforts should be implemented to investigate how this increase may reduce brain impairment associated with neurodegenerative diseases. However, the results here presented have clearly identified some of the synaptic molecular targets of ZnO NPs and that these NPs are worthwhile exploring for their therapeutic potential.O efeito do zinco nos sistemas neuronais está a aumentar o seu interesse na pesquisa científica devido a um possível papel como modulador da atividade sináptica. Além disso, pesquisas recentes apontam para o potencial terapêutico de nanopartículas de óxido de zinco (ZnO NPs) em células neuronais. Essas nanopartículas são excelentes transportadoras de fármacos para o cérebro porque, além de terem características positivas para a proteção da homeostase celular, são pequenas, permitindo que elas ultrapassem a barreira hematoencefálica e interajam diretamente dentro dessas células. Nesse sentido, a presente tese explorou o efeito de concentrações sub-tóxicas de ZnO NPs na viabilidade celular e produção de ROS em células SH-SY5Y, bem como seu impacto nos níveis de expressão das proteínas PSD-95, SHANK 3 e β-actina. Os resultados mostraram aumentos na expressão das proteínas PSD-95 e SHANK 3, sem variações na expressão da β-actina após a exposição das células neuronais a concentrações sub-tóxicas e em tempos de exposição reduzidos aos ZnO NPs. Esforços futuros devem ser implementados para investigar como esse aumento pode reduzir o comprometimento cerebral associado a doenças neurodegenerativas. No entanto, os resultados aqui apresentados identificaram claramente alguns dos alvos moleculares sinápticos das ZnO NPs e que vale a pena explorar o seu potencial terapêutico.Mestrado em Biomedicina Molecula

Designing Advanced Nanocatalysts: Synthesis of complex CeO2-based Nanostructures

[eng] The main objective of this dissertation has been to stablish a bridge between materials science and its fields of application. The scope of this thesis work, under this premise, is aimed towards understanding the catalytical properties of CeO2 nanocrystals and the applications arising from them. In order to tackle it, the chemist labour consists in providing a cutting-edge nanosynthesis technology able to improve efficiencies of the chemical processes, reducing energy consumption and minimizing the environmental impact of both activity and waste products. At the same time, research on nanomaterial synthesis involves the design and formulation of nanomaterials under total control of their physicochemical, morphological, and colloidal properties. Coupled with the appropriate description of the structure-activity relations, the current aim of nanomaterial synthesis is an application-oriented design strategy towards programmable properties of the products. Within this framework, this thesis work is divided into two parts. The first part revolves around nanomaterial synthesis. It pursues the optimised formulation of nanostructured CeO2, a semiconductor material that holds a broad set of intrinsic catalytic properties, describing the synthesis of the minimal stable size for colloidal monocrystalline particles of the material and its complete physicochemical characterization (size, composition, morphology, crystal structure, optical and colloidal properties). It is followed by the extension of the material’s functionality through different derivation strategies, such as doping with different trivalent lanthanide ions and coupling to plasmonic metal domains (Au and Ag) via different synthetic approaches to produce several types of hybrid architectures (core-shell, heterodimers, hollow structures, and other anisotropic shapes) of controlled size. The second part of this work involves the characterization of structure-activity relations of the CeO2-based nanomaterials synthesised in the first part. These are evaluated first through the catalytic performance of each nanomaterial. It has been carried out for two different processes. As a heterogeneous catalyst for methanol production and as ROS scavenger for biomedical applications, coupled with the correspondent assessment of the nanomaterials’ toxicity through in vitro assays. To complete the description of the structure-activity relations, the characterization of the singular electronic structure of CeO2, that confers its characteristic catalytical properties, has been also carried out. Employing core-level spectroscopic techniques, the differences between bulk and nanosized CeO2 have been evaluated through the Ce 3d and O 1s spectra in XPS and Ce L3 edge in XANES.[cat] L'objectiu principal d'aquesta tesi doctoral és fomentar ponts entre la ciència de materials i els seus diferents camps d'aplicació. Partint d'aquesta premissa, la finalitat concreta del treball és assolir uns millor coneixement de les propietats catalítiques del CeO2 nanoestructurat i les aplicacions que en poden emergir. Per abordar aquesta qüestió, des de l'àmbit de la química s'ha de proveir d’una tecnologia capdavantera de síntesi de nanomaterials, capaç de millorar les eficiències dels processos químics reduint el consum d'energia i la producció de residus per minimitzar-ne l'impacte ambiental. Alhora, la investigació actual en síntesi de nanomaterials es fonamenta en el disseny i formulació dels mateixos sota el control total de les seves propietats fisicoquímiques, morfològiques i col·loidals. En conjunt amb la descripció adequada de les relacions activitat-estructura dels materials formulats, l'objectiu actual de la síntesi de nanomaterials és el disseny orientat a la aplicació final mitjançant la programació de les propietats del producte. Dins d'aquest marc, aquesta tesi compta amb dues parts. La primera gira al voltant de la síntesi de nanomaterials. Es busca la formulació optimitzada de nanocristalls de CeO2, un material semiconductor que posseeix un ampli ventall de propietats catalítiques, a través de la descripció de la síntesi de partícules col·loidals monocristal·lines de menor mida possible dins de la seva estabilitat termodinàmica. Aquesta part es completa amb l'extensió de la funcionalitat del material mitjançant diferents estratègies d'hibridació d'aquest, com ara bé el dopatge amb cations trivalents lantànids o l'acoblament de dominis de metalls plasmònics (Au i Ag) seguint diferents estratègies de síntesi. La segona part d'aquest treball tracta la caracterització de les relacions estructura-activitat dels materials ja sintetitzats en la primera. Aquestes són avaluades, en primer lloc, a través de la activitat catalítica de cada nanomaterial per a un procés de catàlisi heterogènia (producció de metanol) i com a neutralitzador de radicals lliures per aplicacions biomèdiques. En segon lloc, l'origen de les propietats catalítiques del material es descriu a través de la caracterització de la singular estructura electrònica del CeO2 nanostructurat, emprant tècniques d'espectroscòpia com XPS i XANES

Genomes and Virulence Factors of Novel Bacterial Pathogens Causing Bleaching Disease in the Marine Red Alga Delisea pulchra

Nautella sp. R11, a member of the marine Roseobacter clade, causes a bleaching disease in the temperate-marine red macroalga, Delisea pulchra. To begin to elucidate the molecular mechanisms underpinning the ability of Nautella sp. R11 to colonize, invade and induce bleaching of D. pulchra, we sequenced and analyzed its genome. The genome encodes several factors such as adhesion mechanisms, systems for the transport of algal metabolites, enzymes that confer resistance to oxidative stress, cytolysins, and global regulatory mechanisms that may allow for the switch of Nautella sp. R11 to a pathogenic lifestyle. Many virulence effectors common in phytopathogenic bacteria are also found in the R11 genome, such as the plant hormone indole acetic acid, cellulose fibrils, succinoglycan and nodulation protein L. Comparative genomics with non-pathogenic Roseobacter strains and a newly identified pathogen, Phaeobacter sp. LSS9, revealed a patchy distribution of putative virulence factors in all genomes, but also led to the identification of a quorum sensing (QS) dependent transcriptional regulator that was unique to pathogenic Roseobacter strains. This observation supports the model that a combination of virulence factors and QS-dependent regulatory mechanisms enables indigenous members of the host alga's epiphytic microbial community to switch to a pathogenic lifestyle, especially under environmental conditions when innate host defence mechanisms are compromised

Kinetoplastid Phylogenomics and Evolution

This Special Issue, Kinetoplastid Phylogenomics and Evolution, unites a series of research and review papers related to kinetoplastid parasites. The diverse topics represented in this collection display a variety of scientific questions and methodological approaches currently used to study these fascinating organisms