Ana A. Ramos

Nutrient Limitation is the Main Regulatory Factor for Carotenoid Accumulation and for Psy and Pds Steady State Transcript Levels in Dunaliella salina (Chlorophyta) Exposed to High Light and Salt Stress

Dunaliella salina (Dunal) Teodoresco (1905) is a green unicellular alga able to withstand severe salt, light, and nutrient stress, adaptations necessary to grow in harsh environments such as salt ponds. In response to such growth conditions, this microalga accumulates high amounts of β-carotene in its single chloroplast. In this study, we show that carotenoid accumulation is consistently inhibited in cells grown in nutrient-supplemented media and exposed either to high-light or medium-low-light conditions. Likewise, carotenogenesis in cells shifted to higher salinity (up to 27% NaCl) under medium-low-light conditions is inhibited by the presence of nutrients. The steady-state levels of transcripts encoding phytoene synthase and phytoene desaturase increased substantially in D. salina cells shifted to high light or high salt under nutrient-limiting conditions, whereas the presence of nutrients inhibited this response. The regulatory effect of nutrient availability on the accumulation of carotenoids and messenger RNA levels of the first two enzymes committed to carotenoid biosynthesis is discussed.

Isolation and characterization of a stress-inducible Dunaliella salina Lcy-β gene encoding a functional lycopene β-cyclase

The halotolerant green alga Dunaliella salina accumulates large amounts of β-carotene when exposed to various stress conditions. Although several studies concerning accumulation and biotechnological production of β-carotene have been published, the molecular basis and regulation of the genes involved in carotenoid biosynthesis in D. salina are still poorly known. In this paper, we report the isolation and regulation of the lycopene β-cyclase (Lcy-β) gene by abiotic stress. The function of this gene was determined by heterologous genetic complementation in E. coli. Gene expression and physiological analyses revealed that D. salina Lcy-β steady-state transcript and carotenoid levels were up-regulated in response to all stress conditions tested (salt, light and nutrient depletion). The results presented here suggest that nutrient availability is a key factor influencing carotenogenesis as well as carotenoid biosynthesis-related gene expression in D. salina.

Characterization of Dunaliella salina strains by flow cytometry: a new approach to select carotenoid hyperproducing strains

In the present work the characterization of different strains of Dunaliella salina from established cell culture collections and various isolates from solar saltworks located in the Canary Island, as well as one mutant, was carried out in order to assess the existence of intraspecific differences and to determine the potential productivity of each one. Morphological characteristics such as cellular size and cellular complexity and parameters linked to cellular physiology, such as pigment content or cellular growth rate, were determined by means of traditional techniques as well as flow cytometry. Results showed a high morphological and physiological intraspecific variability among the studied strains. Results suggest that the application of the lipidic dye Nile red allowed the development of a cytometric method faster than the traditional techniques to select carotenoid hyperproducing strains of microalgae.

Molecular and functional characterization of a cDNA encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase from Dunaliella salina

In green algae, the final step of the plastidial methylerythritol phosphate (MEP) pathway is catalyzed by 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR; EC: 1.17.1.2), an enzyme proposed to play a key role in the regulation of isoprenoid biosynthesis. Here we report the isolation and functional characterization of a 1959-bp Dunaliella salina HDR (DsHDR) cDNA encoding a deduced polypeptide of 474 amino acid residues. Phylogenetic analysis implied a cyanobacterial origin for plant and algal HDR genes. Steady-state DsHDR transcript levels were higher in D. salina cells submitted to nutritional depletion, high salt and/or high light, suggesting that DsHDR may respond to the same environmental cues as genes involved in carotenoid biosynthesis.

Evolution at a Different Pace: Distinctive Phylogenetic Patterns of Cone Snails from Two Ancient Oceanic Archipelagos

Ancient oceanic archipelagos of similar geological age are expected to accrue comparable numbers of endemic lineages with identical life history strategies, especially if the islands exhibit analogous habitats. We tested this hypothesis using marine snails of the genus Conus from the Atlantic archipelagos of Cape Verde and Canary Islands. Together with Azores and Madeira, these archipelagos comprise the Macaronesia biogeographic region and differ remarkably in the diversity of this group. More than 50 endemic Conus species have been described from Cape Verde, whereas prior to this study, only two nonendemic species, including a putative species complex, were thought to occur in the Canary Islands. We combined molecular phylogenetic data and geometric morphometrics with bathymetric and paleoclimatic reconstructions to understand the contrasting diversification patterns found in these regions. Our results suggest that species diversity is even lower than previously thought in the Canary Islands, with the putative species complex corresponding to a single species, Conus guanche. One explanation for the enormous disparity in Conus diversity is that the amount of available habitat may differ, or may have differed in the past due to eustatic (global) sea level changes. Historical bathymetric data, however, indicated that sea level fluctuations since the Miocene have had a similar impact on the available habitat area in both Cape Verde and Canary archipelagos and therefore do not explain this disparity. We suggest that recurrent gene flow between the Canary Islands and West Africa, habitat losses due to intense volcanic activity in combination with unsuccessful colonization of new Conus species from more diverse regions, were all determinant in shaping diversity patterns within the Canarian archipelago. Worldwide Conus species diversity follows the well-established pattern of latitudinal increase of species richness from the poles towards the tropics. However, the eastern Atlantic revealed a striking pattern with two main peaks of Conus species richness in the subtropical area and decreasing diversities toward the tropical western African coast. A Random Forests model using 12 oceanographic variables suggested that sea surface temperature is the main determinant of Conus diversity either at continental scales (eastern Atlantic coast) or in a broader context (worldwide). Other factors such as availability of suitable habitat and reduced salinity due to the influx of large rivers in the tropical area also play an important role in shaping Conus diversity patterns in the western coast of Africa.

A transcriptome resource for the copepod Calanus glacialis across a range of culture temperatures

The copepod Calanus glacialis plays a key role in the Arctic pelagic ecosystem. Despite its ecological importance and ongoing climate changes, limited knowledge at the genomic level has hindered the understanding of the molecular processes underlying environmental stress responses and ecological adaptation. Transcriptome data was generated from an experiment with C. glacialis copepodite (CV) subjected to five different temperatures. We obtained a total of 512,352 high-quality 454 pyrosequencing reads, which were assembled into 55,562 contigs distributed in 128 KEGG pathways. Functional analysis revealed numerous genes related to diverse biological functions and processes, including members of all major conserved signaling pathways. Comparative analysis of acclimated individuals to experimental temperatures has provided information about gene variations observed in several pathways (e.g. genes involved in energy, lipid and amino acid metabolism were shown to be down-regulated with increasing temperatures). These mRNA sequence resources will facilitate further studies on genomics and physiology-driven molecular processes in C. glacialis and related species.

Microsatellite markers for the Arctic copepod Calanus glacialis and cross-amplification with C. finmarchicus

Calanus glacialis is a major component of Arctic zooplankton and a keystone species in Arctic marine ecosystems. Due to the observed climate warming, its numbers are being reduced to the advantage of a sibling Atlantic species Calanus finmarchicus. We developed and characterized the first set of microsatellite markers in this species to investigate its population genetic structure and dispersal capabilities. Nine polymorphic loci displayed an average of 7.3 alleles (range between 2 and 13) and the levels of expected heterozygosity ranged from 0.039 to 0.806. These provide a valuable tool to understand present connectivity patterns across Arctic regions, look for signatures of past climate effects and predict the response to future climate-driven environmental changes. Additionally, due to the cross-amplification with C. finmarchicus, the markers can be used to discriminate between these sibling species.

Wandering behaviour prevents inter and intra oceanic speciation in a coastal pelagic fish

Small pelagic fishes have the ability to disperse over long distances and may present complex evolutionary histories. Here, Old World Anchovies (OWA) were used as a model system to understand genetic patterns and connectivity of fish between the Atlantic and Pacific basins. We surveyed 16 locations worldwide using mtDNA and 8 microsatellite loci for genetic parameters, and mtDNA (cyt b; 16S) and nuclear (RAG1; RAG2) regions for dating major lineage-splitting events within Engraulidae family. The OWA genetic divergences (0–0.4%) are compatible with intra-specific divergence, showing evidence of both ancient and contemporary admixture between the Pacific and Atlantic populations, enhanced by high asymmetrical migration from the Pacific to the Atlantic. The estimated divergence between Atlantic and Pacific anchovies (0.67 [0.53–0.80] Ma) matches a severe drop of sea temperature during the Günz glacial stage of the Pleistocene. Our results support an alternative evolutionary scenario for the OWA, suggesting a coastal migration along south Asia, Middle East and eastern Africa continental platforms, followed by the colonization of the Atlantic via the Cape of the Good Hope.

Biochemistry and molecular biology in Portugal: An overview of past and current contributions

Portugal, represented by the Portuguese Biochemical Society, has been a long-standing member of the IUBMB and a founding member of FEBS. It has organized several international meetings in the broad field of biochemistry and molecular biology, such as the 27th FEBS Meeting (Lisbon, 2001). In 2008, four international conferences on topics ranging from pattern recognition to proteomics are already scheduled to take place in Portugal, showing that the field is dynamic and diverse. Currently, 22 Portuguese higher education institutions (Table 1) offer nine undergraduate and 33 postgraduate programs with a strong emphasis on biochemistry and related fields. Specifically, undergraduate students can select study plans in biochemistry, biotechnology, molecular and cellular biology, genetics, and health sciences. Graduate students can choose from 18 MSc and 15 PhD programs in the aforementioned areas as well as in structural and functional biochemistry, biomedical sciences, computational biology, neurosciences, and molecular genetics. Together with research institutions such as Centre for Neuroscience and Cell Biology of Coimbra (CNC), Institute for Molecular and Cell Biology (IBMC), Gulbenkian Institute of Science (IGC), Institute of Chemical and Biological Technology (ITQB), and many others, they provide a framework for sustainable development of the biochemistry and molecular biology (BMB) field in terms of teaching and research excellence. Here we will review some of the past and present contributions made by researchers in Portuguese scientific institutions to the BMB field. We will address current research trends and we will briefly discuss the opportunities and perspectives presented by them. MATERIALS AND METHODS Most publication and citation records were collected from the ISI Web of Knowledge (WoK) platform using the Science Citation Index Expanded (1900 to present) and the Essential Science Indicators databases. The number of projects approved in scientific areas overlapping the BMB field was collected at Portuguese funding agency (FCT). The list of higher education schools offering undergraduate and graduate programs with a strong emphasis on biochemistry and molecular biology was compiled from the respective institutional websites. This information was later cross-checked with data supplied by Ministry of Science, Technology and Higher Education of Portugal. Most bibliographical references were limited to peerreviewed publications clearly catalogued as belonging to the BMB field. However, this approach excluded important scientific studies that shed light, for instance, on the molecular biology of the cell and metabolism. Therefore, in the sections reviewing specific contributions by scientists in Portuguese institutions, records catalogued as publications in cell biology, endocrinology/metabolism, plant biochemistry, microbiology, and/or neurosciences were included. As electronically stored information is dynamic, retrieval dates are given with reference to this and other information sources.