Poster Session II
3. METATRANSCRIPTOME ANALYSES INDICATE RESOURCE PARTITIONING BETWEEN DIATOMS IN THE FIELD
Harriet Alexander1, 2, Bethany D. Jenkins3, 4, Tatiana A. Rynearson3, and Sonya T. Dyhrman2, 5
1 MIT-WHOI Joint Program in Oceanography/Applied Ocean Science and Engineering, Cambridge, MA 02139, USA
2 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA
3 Graduate School of Oceanography, University of Rhode Island, Narragansett, RI 02882, USA
4 Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA
5 Department of Earth and Environmental Sciences, Lamont-Doherty Earth Observatory, Columbia University, Palisades, NY 10964, USA
The vast diversity of the phytoplankton has long perplexed ecologists, as these organisms coexist in an isotropic environment while competing for the same basic resources. Differential niche partitioning of resources is one hypothesis to explain this “paradox of the plankton,” but it is difficult to quantify and track variation in phytoplankton metabolism in situ. Here quantitative metatranscriptome analyses were used to examine pathways of nitrogen (N) and phosphorus (P) metabolism in diatoms that co-occur regularly in an estuary on the east coast of the US (Narragansett Bay). Expression of known N and P metabolic pathways varied between diatoms, indicating apparent differences in resource utilization capacity that may prevent direct competition. The sampling period spanned a bloom of Skeletonema spp. and highlighted differences in nutrient-related metabolism between Skeletonema spp. and less abundant diatom taxa. Nutrient amendment incubations skewed N:P ratios, elucidating nutrient responsive patterns of expression and facilitating a quantitative comparison between diatoms. Expression of the resource responsive gene set varied over time and differed significantly between diatoms, resulting in orthogonal transcriptional responses to the same environment. Apparent differences in metabolic capacity and the expression of that capacity in the environment suggest that diatom-specific resource partitioning was occurring in Narragansett Bay.
11. COMMUNITY COMPOSITION OF THE MORPHOLOGICALLY CRYPTIC DIATOM GENUS SKELETONEMA IN NARRAGANSETT BAY
Kelly Canesi1 and Tatiana Rynearson2
1 Marine Microbiology Initiative, Gordon and Betty Moore Foundation, Palo Alto, CA; Graduate School of Oceanography, University of Rhode Island, Kingston, RI, USA
2 Graduate School of Oceanography, University of Rhode Island, Kingston, RI, USA
It is well known that morphologically cryptic species are routinely present in planktonic communities but their role in important ecological and biogeochemical processes is poorly understood. We investigated the presence of cryptic species in the genus Skeletonema, an important bloom-forming diatom, using high-throughput genetic sequencing and examined the ecological dynamics of communities relative to environmental conditions. Samples were obtained from the Narragansett Bay Long-Term Plankton Time Series, where Skeletonema spp. can comprise >95% of microplankton cells during blooms. DNA was extracted and sequenced from monthly samples between December 2008 and December 2013, and Skeletonema specific primers were used to exclusively target and differentiate known species. Seven species of Skeletonema were found in Narragansett Bay over the sampling period, including five species that were previously undetected in this location. Skeletonema community composition was highly seasonal, and temperature had the greatest effect on composition changes over time. Winter and spring communities demonstrated lower levels of diversity than did summer and autumn communities. These data suggest that a Skeletonema community can be comprised of different species, even in one estuary, and that seasonal changes appear to have a substantial effect on community structure, and perhaps important ecological processes, over time.
15. TAXON-SPECIFIC RESPONSES OF DIATOM ASSEMBLAGES TO CHANGING CO2 AVAILABILITY IN OYASHIO WATERS OF THE WESTERN NORTH PACIFIC DURING SPRING
Hisashi Endo1, 2, Koji Sugie3, 4, Takeshi Yoshimura4, and Koji Suzuki1, 2
1 Faculty of Environmental Earth Science, Hokkaido University
2 CREST, Japan Science and Technology
3 Research & Development Center for Global Change, Japan Agency for Marine Earth-Science and Technology (JAMSTEC)
4 Central Research Institute of Electric Power Industry
We conducted an on-deck incubation experiment using surface seawater collected from the Oyashio region of the western North Pacific in May 2011 to examine the impact of different CO2 levels on bloom-forming diatom assemblages in the study area. During incubation, the net growth rate of fucoxanthin, a marker pigment for diatoms, was significantly lower in the high (750 and 1000 μatm) and low (180 μatm) CO2 treatments than in the control (350 μatm). Transcript abundance of the diatom-derived rbcL gene, which encodes the large subunit of RuBisCO, also decreased with an increase in seawater CO2 levels. The taxonomic composition and diversity of rbcL gene (DNA) and its transcripts (cDNA) were also examined using Ion Torrent sequencing technology. As a result, in both DNA and cDNA libraries, the diversity of diatoms-derived rbcL decreased in the high CO2 treatments relative to the control, and that was mainly caused by the decrease in relative contributions of Chaetocerotaceae, Thalassiosiraceae, and Fragilariaceae to the total. The present study suggests that the future high-CO2 condition could cause decreases in the biomass, photosynthetic activity, and diversity of bloom-forming diatoms in the Oyashio region during spring.
36. PHYTOPLANKTON ADAPT TO CHANGING OCEAN ENVIRONMENTS
Mount Allison University, Sackville, Canada
Model projections indicate that climate change may dramatically restructure phytoplankton communities with cascading consequences for marine food webs. It is currently not known whether evolutionary change is likely to be able to keep pace with the rate of climate change. For simplicity and in the absence of evidence to the contrary, most model projections assume species have fixed environmental preferences and will not adapt to changing environmental conditions on the century scale. Using 15 years of observations from Station Cariaco, we show that most of the dominant species from a marine phytoplankton community were able to adapt their realized niches to track average increases in water temperature and irradiance, but the majority of species exhibited a fixed niche for nitrate. We don’t know the extent of this adaptive capacity, so we can’t conclude that phytoplankton will be able to adapt to the changes anticipated over the next century, but community ecosystem models can no longer assume that phytoplankton can’t adapt.
44. INTERACTIONS OF DIATOMS AND BACTERIA IN BIOFILMS
Peter G. Kroth, Matthias Buhmann, Birgit Schulze, Miriam Windler, Katrin Leinweber
Fachbereich Biologie, Universität Konstanz, 78457 Konstanz, Germany
Phototrophic, epilithic biofilms are a typical feature of aquatic ecosystems. To study interactions between diatoms and bacteria we isolated and identified different diatoms and associated satellite bacteria. Interestingly, purified diatom cultures showed significant differences with respect to biofilm formation when compared to the corresponding xenic cultures. The diatom Achnanthidium minutissimum forms capsule-like structures in the presence of an isolated bacterial strain, but not in axenic state. SEM analyses show that capsule material that was mechanically stressed by being stretched between or around cells displayed fibrillar substructures. Fibrils were also found on the frustules of non-encapsulated cells, implicating that A. minutissimum capsules may develop from fibrillar precursors. We furthermore have screened the genome of the marine diatom Phaeodactylum tricornutum for gene models encoding proteins exhibiting leucine-rich repeat (LRR) structures. We were able to identify several transmembrane LRR-proteins, which are likely to function as receptor-like molecules. Moreover, P. tricornutum encodes a family of secreted LRR proteins likely to function as adhesion or binding proteins as part of the extracellular matrix. We furthermore have analyzed the extracellular proteome of P. tricornutum in the presence and absence of Roseovarius bacteria.
51. CHANGES IN POLYAMINE POOLS DURING GROWTH OF THALASSIOSIRA PSEUDONANA: RESPONSE TO ENVIRONMENTAL STRESS AND NUTRIENT LIMITATION
Qian Liu1, Naoyoshi Nishibori2, Ichiro Imai3 and James T. Hollibaugh1
1 Department of Marine Sciences, University of Georgia, Athens Georgia 30602-3636, USA
2 Shikoku University Junior College, Shikoku University, Ojin, Tokushima 771-1192, Japan
3 Laboratory of Marine Biology, Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minatomachi, Hakodate, Hokkaido 041-8611, Japan
Polyamines serve diverse functions in eukaryotes. In diatoms, long-chain polyamines control silica precipitation during formation of the cell wall. We found 1, 3-diaminopropane, putrescine, spermidine, nor-spermidine and nor-spermine in polyamine pools extracted of Thalassiosira pseudonana, while spermine was not detected. Combining these observations with previous studies (Bagga et al.1991; Armbrust et al. 2004), we hypothesize that polyamine biosynthesis in T. pseudonana occurs as follows: putrescine is synthesized from ornithine, an intermediate of the urea cycle; and then produces spermidine, mediated by spermidine and spermine synthases. Oxidation of spermidine to 1, 3-diaminopropane by a spermine synthase orthologue produces nor-spermine, with nor-spermidine produced as an intermediate.
We also investigated effects of growth stage and environmental conditions on extractable polyamine pools of T. pseudonana. We found that polyamines, especially putrescine, accumulate from lag to late stationary phase and in response to increasing temperature or decreasing salinity. Polyamines released to the medium were coupled to the composition of intracellular pools. Increasing the concentration of limiting nutrients (N or P) in the growth medium expanded the polyamine pool, but raising Si concentration resulted in decreased pool sizes. Polyamines tended to be released to the medium under low nutrient conditions.
52. DECIPHERING THE MECHANISMS BEHIND CLIMATE-DRIVEN CHANGES IN THE RELATIVE ABUNDANCES OF CYCLOTELLA SENSU LATO TAXA
Heera I. Malik & Jasmine E. Saros
Climate Change Institute and School of Biology and Ecology, University of Maine, Orono, ME, USA
The widespread increase in the relative abundances of Cyclotella taxa in artic and alpine lakes have been attributed to either direct or indirect effects of temperature. The primary objective of this study was to investigate the interactive effects of temperature, light, and nutrients on Cyclotella abundances and growth rates. To do so, we conducted 2x2x2 factorial design in-lake experiments with temperature, light, and nutrients in two lakes: one with phytoplankton assemblages dominated by diatoms, and one dominated by other phyla. The secondary objective was to determine whether the structure of phytoplankton communities alters the response of Discostella stelligera to abiotic factors. We observed that each species responded differently under each treatment condition. Interactive effects of nutrients-light-temperature effected cell densities of Puncticulata bodanica, Cyclotella comensis, Cyclotella ocellata, and Discostella stelligera whereas Puncticulata radiosa was affected by light and nutrients only. Discostella stelligera responded differently to temperature in the two different communities, with higher temperatures reducing its relative abundance when it occurred in the mixed community. This study will provide more ecological information for key species to improve our ability to assess non-synchronous change in diatom communities across lakes.
56. BACTERIAL COMMUNITY PROFILE OF PHAEODACTYLUM TRICORNUTUM CULTURES
Fiona Wanjiku Moejes1, 2, Ovidiu Popa2, Julie Maguire1 and Oliver Ebenhöh2
1 Daithi O’Murchu Marine Research Station, Gearhies, Bantry, Co. Cork, Ireland
2 Institut für Quantitative und Theoretische Biologie, Heinrich-Heine-Universität, Universitätsstr. 1, 40225 Düsseldorf, Germany
The manufacture of microalgal-derived products requires growing microalgal cultures on a commercial scale. A significant obstacle is cultural contamination, especially by bacteria. But is contamination only a negative phenomenon? We are surrounded by examples of interspecies cooperation, and mutualistic symbiosis can potentially enhance growth and productivity . Bacteria and diatoms have coexisted in habitats throughout the oceans for more than 200 million years , and this PhD project aims to gain insight into this relationship by identifying the bacterial consortia in laboratory-scale 5L Phaeodactylum tricornutum cultures, and follow the community over time and in either complete or minimal culture media. The bacterial community profile was constructed using barcoded-16S rRNA pyrosequencing, following a pipeline to filter the reads, and matching them to species in a reference database. A modified version of PermanovaG was used to carry out permutational multivariate analysis of variance, and showed statistically significant shifts in the bacterial community over time, and in differing media. Initial results show three dominant bacterial families; Pseudoalteromonadaceae, Alteromonadaceae and Flavobacteriaceae, which all demonstrate interesting shifts in abundance over time and in differing media. Using literature, we can hypothesize on the varying roles of the bacterial community. Future plans are to verify them experimentally.
 Kazamia E; Aldridge DC; Smith AG, 2012. Synthetic ecology – a way forward for sustainable algal biofuel production? Journal of Biotechnology 162: 163-169
 Amin SA; Parker MS; Armbrust EV, 2012. Interactions between diatoms and bacteria. Microbiol. Mol. Biol. Rev. 76(3): 667-684
The project is one of 14 projects that make up AccliPhot, a European Commission Seventh Framework Project where the ultimate scientific goal is to establish a systems-wide understanding of the acclimation processes of the photosynthetic machinery in microalgae (green algae and diatoms) and higher plants.
68. CHARACTERIZING DIATOM BIODIVERSITY IN THE NORTH PACIFIC USING 18S TAG SEQUENCING
Diane M. Rico, E. Virginia Armbrust
School of Oceanography, University of Washington, Seattle, WA, USA
Because diatoms and other eukaryotic phytoplankton are important primary producers that facilitate biogeochemical cycling and carbon export, their community structure can profoundly impact the functioning of marine ecosystems. Although diatom populations differ across oceanographic regions, few studies have assessed how their species composition and biodiversity are affected by environmental and latitudinal gradients. Here, we use 18S tag sequencing to assess community structure in eukaryotic phytoplankton along a cruise track in the North Pacific extending from Seattle, Washington to Station ALOHA, north of Hawaii. The transect extends through several oceanographic regions with marked differences in nutrient concentration, temperature, and salinity. Samples were taken from surface and chlorophyll maximum depths at each of 10 stations along the cruise track, and eukaryotes in the 1.6 μm – 53 μm size fractions were analyzed in this study. Preliminary data show a general increase in diatom species richness with increasing latitude. Estimates of unique species at the chlorophyll maximum depths range from 34 to 21 at coastal stations, decrease to 12 at the transition zone, and only two unique species were detected at Station ALOHA. Surface sampling revealed 53 estimated species near the coast, and less than seven species at stations south of the transition zone.
82. INTERACTIONS BETWEEN DIATOMS AND AN ANTAGONISTIC FLAVOBACTERIUM
Helena M. van Tol1, Shady A. Amin1,2, E. Virginia Armbrust1 1 School of Oceanography, University of Washington, Seattle, Washington 98195, USA
2 Chemistry Faculty, New York University Abu Dhabi, PO Box 129188, Abu Dhabi, United Arab Emirates
Diatom physiology is influenced by a consortium of heterotrophic bacteria that consume the products of photosynthesis. Bacteria that concentrate within the diatom cell’s diffusive boundary layer impact the cell’s immediate environment by producing metabolites, consuming metabolites produced by the diatom and other bacteria, as well as competing for and regenerating inorganic nutrients. We found that the flavobacterium Croceibacter atlanticus is consistently associated with the diatom Pseudo-nitzschia multiseries in culture. Co-culture experiments and epifluorescence microscopy showed that Croceibacter attaches to the cell surface of diatoms and negatively impacts the growth a wide range of heterokonts including P. multiseries, P. fraudulenta, Thalassiosira oceanica, T. pseudonana, and Aureococcus anophagefferens. When co-cultured with Croceibacter, we found that T. pseudonana elongates, forms aggregates, and produces large globular chloroplasts. Cell cycle analysis with flow cytometry indicated that Croceibacter arrests T. pseudonana in the G2 phase of mitosis. We hypothesize that Croceibacter inhibits cellular division in diatoms while chloroplasts continue replicating, possibly enhancing photosynthesis and leakage of metabolites into the media.
85. DIATOMS IN SPACE: GLOBAL POPULATION STRUCTURE AND ENVIRONMENTAL SELECTION IN THE MARINE DIATOM T. ROTULA
Kerry Whittaker and Tatiana Rynearson
Graduate School of Oceanography, University of Rhode Island, Narragansett, RI, USA
Diatoms exhibit astounding levels of diversity, yet the mechanisms driving their divergence are little understood. We determined global population structure among over 400 isolates of the ecologically important diatom species Thalassiosira rotula. Isolates were sampled at five time points over 12 months at sites distributed across the global ocean and across a wide range of environmental conditions. Isolates were genotyped using six microsatellite markers developed from a partial T. rotula genome sequence. Samples exhibited enormous diversity; within-sample clonal diversity was as high as 100%, and between-sample FST values were upwards of 0.2. The presence of genetically distinct populations demonstrates that significant levels of genetic isolation can occur in the ocean despite the enormous dispersal potential of these planktonic organisms. Principle coordinates analysis (PCA) and isolation-by-distance measures demonstrated that genetic distance was unrelated to geographic distance; this suggests a high potential for global dispersal, and that gene flow in this species is not limited by distance. The presence of significantly diverged populations correlated strongly with ecological variables including temperature, suggesting that environmental selection plays a much larger role than dispersal in structuring the distribution of T. rotula populations across the globe.
97. MOLECULAR DIVERSITY OF BENTHIC DIATOMS IN INTERTIDAL MUDFLATS OF THE WEST COAST OF KOREA
Eun Chan Yang, Sung Min An, Jae Hoon Noh and Dong Han Choi
Korea Institute of Ocean Science & Technology, Ansan 426-774, Korea
Identification and classification of benthic diatoms are one of the most challengeable tasks because of its morphological similarity and complexity with small size of cell. In present study, we isolated and developed 64 strains of benthic diatom from several sampling stations of intertidal mudflat, in the west coast of Korea. The new strains were represented 56 species of 14 genera, and subjected to molecular analyses using two different genes (i.e., rbcL and 18S rDNA), as well as various morphological analyses. Most of strains belong to the Bacillariaceae (23 strains) and Naviculaceae (21 strains) family groups, including 20 newly identified species. The rbcL and 18S rDNA sequences were useful to identifying 8 and 12 strains, respectively. Genetic distances (P-distance) of rbcL and 18S rDNA compared in the genus and family levels of each group. In the nitzschoid diatom group, Bacillaria and Cylindrotheca were monophyletic, however, Nitzshia and Tryblionella were polyphyletic. Naviculoid diatoms group includes monophyletic Haslea, Gyrosigma, Pleurosigma and Navicula. Two tube dwelling diatoms (i.e. Berkeleya and Parlibellus) were grouped within asymmetrical biraphid diatoms comprising of the genus Entomoneis and Petrodictyon. These results will useful for better understandings on molecular and morphological diversity studies of benthic diatoms.
2. POPULATION STRUCTURE OF ECOLOGICALLY SIGNIFICANT DIATOMS IN SOUTH EASTERN AUSTRALIA USING MICROSATELLITE MARKERS
Penelope Ajani1, Gustaaf Hallegraeff2, Gurjeet Kohli1 and Shauna Murray1
1 Plant Functional Biology and Climate Change Cluster (C3), University of Technology, Sydney , PO Box 123, Broadway NSW 2007, Australia, and Sydney Institute of Marine Science, Chowder Bay Rd, Mosman NSW 2088, Australia
2 Institute for Marine and Antarctic Studies, University of Tasmania, Private Bag 129, Hobart, TAS 7001, Australia
Diatoms are common in well mixed, coastal upwelling regions across the global ocean. The East Australian Current (EAC) is the dominant western boundary current in eastern Australia and is important in controlling phytoplankton productivity and distribution. The influence of the EAC on the connectivity of marine phytoplankton populations in this region is as yet unknown, however. This study will use microsatellite markers to characterise the population biology and connectivity of the ecologically significant diatom Leptocylindrus danicus along the east coast of Australia. Culture studies under future climate change scenarios should identify key environmental stressors on these species. This information will assist in determining the diversity of ecotypes present and help to predict the impacts of the strengthening of the EAC on critical coastal food chains.
6. RAD RESULTS – USING POPULATION GENOMICS TO INVESTIGATE THE STRUCTURE OF ENDEMIC DIATOM POPULATIONS IN LAKE BAIKAL, SIBERIA
Mariska C. Brady1 and Edward C. Theriot2
1Section of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA
2Texas Natural Science Center, University of Texas at Austin, Austin, TX 78712, USA<
We know a great deal about Lake Baikal, Siberia. We know how old it is, how deep, how voluminous, how cold. We know how frequently Lake Baikal’s endemic phytoplankton bloom, and we have data on the community response to such events. Lake Baikal offers a natural experiment in which to examine the potential response of phytoplankton to documented warming in the lake. With three quasi-independent basins along a 636 km N-S axis, and only modest water exchange among these basins, there is the potential for genetic differentiation of planktonic diatoms at a finer scale. In particular, we set out to determine how populations within endemic diatom species are distributed in the world’s oldest, deepest, most voluminous lake. To answer this question, we employed ddRAD (Double Digest Restriction-site Associated DNA) sequencing and population genomics to characterize population genetic statistics of Aulacoseira baicalensis, while testing specific temporal and geographic hypotheses about the population structure of this charismatic, endemic diatom. The results of these analyses will be discussed, along with the unique challenges of applying this technology to “difficult-to-culture” diatoms from the other side of the world.
7. AN INTEGRATED ANALYSIS OF NITROGEN AND PHOSPHORUS DEPRIVATION RESPONSES IN PHAEODACTYLUM TRICORNUTUM
Leila Alipanah, Jens Rohloff, Per Winge,Javad Najafi, Tore Brembu and Atle M. Bones
Department of Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway
Nitrogen (N) and phosphorus (P) are essential elements for all living organisms, and changes in N or P availability in aquatic systems affect diatom growth and productivity. We investigated the adaptive mechanisms in Phaeodactylum tricornutum to N and P deprivation using a combination of transcriptomics, metabolomics, and physiological and biochemical experiments. Photosynthetic capacity and chlorophyll content of the cells decreased during both N and P deprivation; a majority of the genes associated with photosynthesis and chlorophyll biosynthesis were also repressed. The strongest induction of gene expression was observed for proteins involved in N and P acquisition and scavenging. Carbon metabolism was restructured during both stresses through down-regulation of the Calvin cycle and chrysolaminarin biosynthesis and up-regulation of chrysolaminarin degradation, cytosolic glycolysis and the oxidative pentose phosphate pathway. P deprivation also resulted in alterations of the reductive part of pentose phosphate pathway. Transcriptome data indicated that membrane lipids contributed considerably to triacylglycerol accumulation during both treatments through conversion by phospholipases. In P-deprived cells, the induced expression of genes encoding sulfolipid biosynthesis enzymes and a putative betaine lipid biosynthesis enzyme indicated replacement of phospholipids with non-P lipids.
9. LEVERAGING RNASEQ FOR GENE MODEL IMPROVEMENT IN THALASSIOSIRA PSEUDONANA
Raffaela Abbriano Burke1, David Lopez2, Jing Lu2, Jesse Traller1, Roshan Shrestha1, Aubrey Davis1, Sean Gallaher3, Michael Thompson2, Marco Morselli2, Matteo Pellegrini2, Mark Hildebrand1
1Marine Biology Research Division, Scripps Institution of Oceanography, UC San Diego, La Jolla, CA USA
2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA, USA
3Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA USA
Our current understanding of the organization and regulation of metabolism in diatoms relies heavily on availability of genomic information and accurate gene model annotation. The publication of the Thalassiosira pseudonana genome as a model photosynthetic eukaryote greatly expanded our understanding of the genetic basis for the unique aspects of diatom biology. However, many of the Thaps3 gene predictions are incorrect, obscuring important information such as subcellular targeting signals. High-throughput RNAseq reads can provide valuable information for gene model improvement. We combined existing models with evidence from a high coverage RNAseq dataset to inform new gene model predictions using the AUGUSTUS algorithm. Over half of the 13,399 genes predicted are modified from the Thaps3 reference, including revised gene boundaries, novel splice sites, and unique loci containing 1,300 previously unannotated genes. Representative models involved in diatom primary metabolism were confirmed using RT-PCR and/or chosen for in-vivo localization studies to validate targeting predictions. Additionally, we generated a downloadable GFF file to easily visualize the models through the IGV genome browser to facilitate the use of these models as a resource to inform future transcriptomic, comparative genomic and physiological studies.
12. LIGHT-DEPENDENT TRANSCRIPTIONAL REGULATION IN DIATOMS.
Sacha Coesel1, Justin Ashworth1, E. Virginia Armbrust2, Mónica V. Orellana 1,3 and Nitin Baliga 1,4
1 Institute for Systems Biology, Seattle WA USA
2 School of Oceanography, University of Washington, Seattle WA USA
3 Polar Science Center-APL, University of Washington, Seattle WA USA
4 Department of Microbiology, University of Washington, Seattle WA USA
Light, the fundamental source of energy for photosynthetic organisms, is a highly fluctuating environmental factor for marine species. Most organisms possess photoreceptors to perceive light signals necessary for orchestrating basic diel metabolic processes and adapting to changing light conditions. Diatoms possess photoreceptors from the cryptochrome and phytochrome families. Their genomes also encode unique heterokont-specific photoreceptors, aureochromes, able to bind DNA when activated by light. These latter proteins have the potential to control transcriptional regulation without using intrinsic intercellular signal transduction cascades. The Phaeodactylum tricornutum Aureo1A protein has recently been shown to bind and activate the promoter region of a diatom-specific cyclin controlling the light-dependent G1-to-S cell cycle checkpoint. The regulatory functions of other aureochromes are still unknown. In this work, we mined over 20 phylogenetically diverse diatom species for known and novel light regulatory-encoding sequences using InterProScan-based searches. We found expanded and species-specific putative light-sensitive elements, indicating that light may act as a strong selective force on diatom species differentiation. We have explored putative roles for photoreceptors and other light-responsive regulatory elements by analyzing their light condition-dependent co-regulation with all genes in the genome of P. tricornutum and Thalassiosira pseudonana. Insights revealed by these analyses will be discussed at the meeting.
20. MORPHOLOGICALLY CRYPTIC SPECIES AND PHYLOGEOGRAPHIC PATTERNS IN THE DIATOM GENUS THALASSIOSIRA
Gang Chen, Tatiana A. Rynearson
Graduate School of Oceanography, University of Rhode Island, Narragansett, RI 02882, USA
Morphologically cryptic diatom species are being increasingly identified in field samples reflecting the high level of species diversity present within the diatoms. We collected cell isolate DNA samples from a global distribution range for two morphologically indistinguishable species Thalassiosira gravida/rotula. By using three molecular genetic markers (nuclear 18S, ITS, and mtCOII), we identified the two target species along with a number of co-occurring Thalassiosira species, including T. aestivalis, T. nordenskioldii, T. anguste-lineata, T. miniscula. The newly developed mitochondrial gene marker, cytochrome c oxidase subunit II (mtCOII) showed a general genealogical concordance with 18S and ITS, but higher inter- and intraspecific diversity. The sequence divergence of mtCOII was ~10% among those commonly occurring Thalassiosira species, while ~4% between two young sibling species of T. gravida and T. rotula. Intraspecific variation at mtCOII was remarkable in T. gravida, with 10 recorded haplotypes forming two well-diverged geographic clades (~2%) between North Atlantic and Northeast Pacific, suggesting geographic isolation and/or local adaptation may contribute to its genetic structure at the global scale. In North Atlantic, geographic populations (Gulf of Maine, Disko Bay, Icelandic basin) were dominated by one haplotype with derived private ones, suggesting a shared phylogeographic origin, strong gene flow, and some level of differentiation among local populations.
23. LIFTING THE IRON CURTAIN: PHYLOGENETIC COMPARISON OF IRON RESPONSIVE-GENES ACROSS DIATOM SPECIES
Kristofer M. Gomes1, Bethany D. Jenkins1,2
1Department of Cell and Molecular Biology and 2Graduate School of Oceanography, University of Rhode Island, Kingston, RI, 02881, USA
Within marine environments primary production can be limited in large regions of the ocean by low iron availability, including regions characterized as high nutrient low chlorophyll (HNLC). This low iron induced limitation of primary production is due to the requirement of iron in components within essential metabolic pathways including photosynthesis, mitochondrial electron transport, and nitrate assimilation. Diatoms are important primary producers within marine environments and can bloom and accumulate high biomass during introduction of iron into low iron waters despite prebloom low cell density. These observations indicate that diatom species have adaptations that allow for their survival in iron limited waters and rapid growth when iron becomes more abundant. While these adaptations are apparent, their genetic and functional roles are unclear. A large portion of genes identified as sensitive to iron limitation in transcriptome profiling experiments encode proteins of unknown function. In order to begin to elucidate a proposed function for these genes active in iron limited responses a bioinformatics approach was taken using homology searching of known iron stress response proteins against a database of over seventy diatom species’ transcriptomes. Phylogenetic analysis was conducted to better understand how these genes are distributed across diatom lineages.
28. A YEAST ONE-HYBRID SCREENING PLATFORM TO UNRAVEL UV-B TRANSCRIPTIONAL REGULATION IN PHAEODACTYLUM TRICORNUTUM
Marc Heijde, Hilde Van den Daele, Lieven De Veylder
Dept of Plant Systems Biology, VIB, University of Ghent, Belgium
UV-B irradiance which is not completely absorbed by the stratospheric ozone layer and is a major factor to consider in the climate change context, has a negative impact on primary producers by reducing the photosynthetic uptake of atmospheric carbon dioxide and consequently affecting species diversity, ecosystem stability, trophic interactions and global biogeochemical cycles. Photosynthetic organisms protect themselves from potential UV-B caused damages by producing UV-B absorbing compounds and by activating DNA repair. In plants, these UV-B responses are under control of a UV-B specific signaling pathway not conserved in marine phytoplankton. However, diatoms also show the ability to acclimate and to develop long term responses to UV-B but underlying mechanisms are still largely unknown. In the presented research, we are deciphering the transcriptional networks involved in UV-B signalling by identifying the upstream transcriptional regulators of UV-B regulated genes. Using Phaeodactylum tricornutum, we developed a diatom transcription factor collection (circa 200 genes) and set up a semi-automated yeast one-hybrid platform to identify the transcription factors responsible for the UV-B transcriptome reprogramming in diatoms.
94. GENOMIC PHASING SUPPORTS LOSS OF SEX IN 5 OF 7 THALASSIOSIRA PSEUDONANA ISOLATES
Nao Hiranuma1, Tony Chiang1,2, Chris Berthiaume2, Walter L. Ruzzo1,3,4 & E. Virginia Armbrust2
1 University of Washington, Computer Science and Engineering, WA, USA 98195‐2350, USA
2 University of Washington, School of Oceanography, Seattle, WA, USA 98195‐7940, USA
3 University of Washington, School of Medicine, Genome Sciences, WA, USA, 98195‐2350, USA
4 Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA, 98102, USA
The diploid diatom Thalassiosira pseudonana has never been observed to reproduce sexually in culture. Chiang, et al. (this conference) have shown that a group of 5 globally dispersed T. pseudonana isolates (CCMP 1335, 1007, 1012, 1014, 1015) exhibit extreme divergence from Hardy‐Weinberg equilibrium, while two others (1013, 3367) do not, likely meaning that the former lack the ability to sexually reproduce not only in culture but also in wild, while the latter retain sex. To independently support this hypothesis, using the GATK genome analysis package we have (partially) phased single nucleotide polymorphisms (SNPs) in each isolate. I.e., for many groups of SNPs we have deduced which variants are co‐resident on each chromosome. Using this, we estimated the density of crossover events per kilobase between pairs of these genomes. Preliminary analysis shows that crossover events are at least 5~7 fold more frequent when comparing to either of the (presumed) sexually reproducing isolates than when comparing any pair of the (presumed) asexuals, strongly consistent with the model positing that the 5 isolates represent a single lineage in which sexual reproduction has been lost, while the other two retain meiotic recombination.
31. REGULATION OF SILICA METABOLISM IN THE DIATOM FRAGILARIOPSIS CYLINDRUS
Amanda Hopes & Thomas Mock
School of Environmental Sciences, University of East Anglia, Norwich, UK
The silica frustule is the most characteristic feature of diatoms with strong implications for their success, diversity and global importance. Sequencing of the polar diatom Fragilariopsis cylindrus (FC) has led to the discovery of a gene containing both a transcription-associated Myb domain and a domain with homology to a silicon transporter (SIT).This is the first diatom gene potentially involved in regulating genes that contribute to synthesising diatom frustules. This discovery opens an unprecedented opportunity to study genetic networks underpinning silica frustules. A transformation system has been developed in FC, with zeocin resistance and egfp run on endogenous fcp promoters. This is currently being used to determine the function of the SIT/Myb gene, following gene modelling. A CRISPR-Cas system is also being developed for knock-outs in T.pseudonana. Modelling of the SIT/Myb gene shows a high confidence Myb domain, a helical SIT domain and possible silica binding motifs. Allelic variants show 100% conservation within both domains and a highly conserved 3’ end. This may be an important starting point for determining how genes involved in silica metabolism are regulated. Development of a FC transformation system allows the use of overexpression, knock-downs and knock-outs, which are invaluable for elucidating the function of any FC gene or pathway.
43. WHAT IS THE SIT-UATION WITH DIATOMS?
Julie A. Koester1, Colleen A. Durkin2, Sara J. Bender3, and E. Virginia Armbrust4
1 University of North Carolina Wilmington, USA
2 Moss Landing Marine Laboratories, USA
3 Woods Hole Oceanographic Institution, USA
4 University of Washington, USA
In the modern ocean, diatoms control the cycling of silicon, but the how, why, and when of this control remain the subject of much inquiry. Silicon cycling is partially controlled by silicic acid transporter (SIT) proteins that actively transport silicic acid into the cell. In this study, aphylogenetic tree was created to explore SIT divergence and diversity and included SIT genes from diatoms and SIT homologs from other protists. The number of known SIT sequences was doubled by including sequences from the Marine Microbial Eukaryote Transcriptome Sequencing database. Interestingly, individual diatom species often encoded SIT genes from multiple distinct clades. SIT-like sequences were detected in additional non-diatom species, raising the possibility for an ancestral SIT clade shared among diatoms and related protists. To determine the ecological relevance of the SIT clades, metatranscriptome Illumina reads collected from surface stations along Line P in the northeast Pacific, were mapped onto the expanded SIT tree. The combined phylogenetic and environmental expression patterns suggest a role for subfunctionalization among SIT proteins and across diatom lineages – features that likely play an important role in how diatom communities take up silicon under changing environmental conditions.
95. UNCOVERING THE EVOLUTIONARY HISTORY OF PSEUDO-NITZSCHIA: CONNECTING PHYSIOLOGY AND MOLECULAR PHYLOGENY
Terence Leach1, Michele Guannel2, Michael Carlson1 and Gabrielle Rocap1
1 University of Washington School of Oceanography, Seattle, WA, USA
2 C-MORE at University of Hawaii, HI, USA
Pseudo-nitzschia is a diverse and cosmopolitan genus of diatoms composed of 37 species and known for its production of the neurotoxin domoic acid (DA). The phylogeny of the Pseudonitzschia genus is complex because it contains many cryptic species complexes and new species are discovered frequently. We sought to evaluate the phylogeny of Pseudo-nitzschia using both ribosomal LSU sequences and the rbcL gene, which encodes the large subunit of the carbon fixation enzyme, ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) in order to better understand the evolutionary relationships within the genus. Trees based on rbcL sequences provided better resolution to resolve the branching pattern of Pseudo-nitzschia species and supported the presence of two clades within the genus. Each clade had distinct morphological and physiological characteristics, such as cell size, frustule morphology, pigment content and DA production, which further supported these two clades. Metatranscriptomic reads from four stations from a transect off the Washington coast to the open ocean were placed on the rbcL trees and reflected a biogeographical distinction between the two clades. The phylogeny of the rbcL shows that it is an excellent marker for both phylogenic comparison as well as community profiling in the environment.
46. GENOME-SCALE MODEL RECONSTRUCTION FOR THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM
Jennifer Levering1, Jared Broddrick1, Alessandra A Gallina1,2, Christopher L. Dupont3, Andrew E Allen3,4, Bernhard Ø Palsson1 , Karsten Zengler1
1 Bioengineering Department, University of California San Diego, La Jolla, CA, USA
2 Colorado State University, Fort Collins, CO, USA
3 J. Craig Venter Institute, La Jolla, CA, USA
4 Integrative Oceanography Division, Scripps Institute of Oceanography, University of California San Diego, La Jolla, CA, USA
Genome-scale metabolic models are fundamental for the analysis of cellular processes at a systems level and represent an organizational framework for analyses of functional genomics, experimental work and computational studies. Based on the genome and manually curated reference models we generated a draft metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Using several resources, such as organism-specific literature, experimental data and pathway databases we manually curated each pathway. The reconstructed metabolic network accounts for more than 1,000 genes associated with approximately 4,300 reactions and about 2,000 metabolites distributed across six compartments, namely cytoplasm, extracellular space, chloroplast including thylakoid lumen, mitochondria and peroxisome. The reactions are mass and charge balanced and roughly 93% of the reactions have associated genereaction rules. Different identifiers such as KEGG reaction and compound IDs were used to annotate reactions and metabolites and enable the comparison to metabolic networks of other microalgae. The model was evaluated and tested against well-known metabolic capabilities of P. tricornutum such as growth on different carbon and nitrogen sources. The model is applied to gain insight into light-driven lipid metabolism in P. tricornutum and to predict strategies to optimize P. tricornutum's metabolism for next-generation biofuel production.
49. GENETIC AND PHYSIOLOGICAL RESPONSES OF THE MODEL DIATOM PHAEODACTYLUM TRICORNUTUM TO LONG TERM OCEAN ACIDIFICATION
Xin LIN, Ruiping HUANG, Futian LI, Yahe LI, Kunshao GAO
State Key Laboratory of Marine Science, Xiamen University, Xiamen, Fujian Province, China
Ocean acidification (OA) which is caused by increasing anthropogenic carbon dioxide CO2, poses new ecological pressures on the whole marine ecosystem. The diatoms contribute 40% of oceanic primary production and play important roles in the marine ecosystem. Many studies investigating impacts of OA on diatoms have been reported. Most studies focus on the responses of diatoms to short term (8-25 generations) OA. Though important information has been obtained from these short-term acclimation experiments, Long-term adaptation experiments are indispensable for investigating the evolutionary responses of diatoms to OA and the underlying mechanisms. Herein, the Phaeodactylum tricornutum strain isolated from South China Sea has been grown at 380 μatm and 1000 μatm for about 2000 generations. For the cells grown under two CO2 conditions, the physiological performance, such as photosynthesis performance and carbon assimilation ability, were measured. In parallel, the transcriptomes for exploring gene expression patterns were conducted as well. In this presentation, we will focus on analyses of the physiological and genetic orchestra associated with the cells state under the two CO2 conditions.
50. DINOFLAGELLATES VERSUS DIATOMS: GENOMIC VIEWS ON THEIR ENERGY AND NUTRIENT NICHES
Department of Marine Sciences, University of Connecticut, Groton, CT 06340, USA
While diatoms generally dominate major coastal marine plankton communities and champion spring blooms in temperate regions, dinoflagellates are as widespread and often take over the system in the form of harmful algal blooms. Diatoms rely on silicate and generally prefer cold nutrient rich waters while dinoflagellates feature motility associated with flagella and nutrient versatility due to mixotrophic/heterotrophic capability. Yet these obvious features do not seem to explain well how the two rivals play their competitive roles in the coastal arena. This presentation is aimed at providing a genomic perspective on their respective unique genetic potentials in harnessing energy and acquiring different forms of nutrients. How Si uptake ability might have come and gone between the two lineages will be discussed.
54. THE KNOCKOUT OF THE NITRATE REDUCTASE GENE CAUSES VAST PHYSIOLOGICAL AND BIOCHEMICAL TRANSFORMATIONS OF PHAEODACTYLUM TRICORNUTUM.
James K. McCarthy, Hong Zheng, Andrew A. Allen
J. Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA 92037
A knockout of the nitrate reductase (NR) gene and the subsequent absence of the NR protein in Phaeodactylum tricornutum cells was produced by transforming P. tricornutum with two plasmids simultaneously. One plasmid consisted of a TALEN with an endonuclease fused to a binding-domain sequence targeting a specific region of the NR gene; the second contained an antibiotic resistance cassette flanked by regions of the NR gene upstream and downstream of the TALEN target site. The initial positive transformants were verified by PCR, phenotypic growth assays and western blots. The PCR results show the antibiotic cassette incorporated into the NR gene and only within the NR loci in the genome. A growth comparison with NR-KO cells lines and WT cultures grown in F/2 amended media either with ammonium or nitrate, and repeated over many generations, shows NR-KO cells growing on ammonium, but not growing on nitrate. Westerns blots of a protein samples gathered for a time-course, RNA-seq analysis of the NR-KO lines vs WT show no NR protein in the knockout for the time course. Subsequent investigations of the knockouts by confocal microscopy and freeze fracture SEMs show a dramatic reorganization of normal pennate diatom physiology. KO cells are swollen and deformed with chloroplasts reduced and pushed to the sides or ends of the cells. Analysis of nitrate remaining in the media and nitrate present in the cells, measured on a per-cell basis, over a seven day time-course, indicates that after 3 days nitrate concentrations in the KO cells rise above 1M (the experiment began with 300 uM nitrate), whereas all nitrate has been assimilated into WT cells at the same time point.
FTIR results indicate a significant rearrangement of protein and carbohydrate fractions in the scans of the WT and NR-KOs samples; an increase over a very short time frame is also observed in the NR-KO lipid fraction as compared to the wild type. FAME analysis of cell lipids shows a profound redistribution of the fatty acid profile in the knockout line. The RNA-seq experimental data, considered broadly, confirms the physiological and biochemical transformation of P. tricornutum caused by the loss of function of nitrate reductase; the details of which are too great to describe in the abstract.
64. METABOLIC LABELING OF RNA OPENS UP NEW HORIZONS IN DIATOM RESEARCH
Minoli Perera and Deborah L. Robertson
Clark University, Biology Department, 950 Main Street Worcester, MA
Cellular RNA levels represent the integration of RNA synthesis, processing, and degradation. In vivo metabolic labeling can be used to measure both global and gene-specific RNA transcription and degradation rates. In addition, it is a valuable tool for exploring RNA:protein interactions. The technique uses sulfur-substituted molecules that are rapidly taken up by cells and incorporated into RNA. The Thalassiosira pseudonana genome encodes both uracil phophoribosyltransferase (UPRT) and uridine kinase (UK), which allow for the incorporation of 4-thiouracil (4tU) and 4-thiouridine (4sU), respectively. Both 4tU and 4sU were detected in T. pseudonana RNA after a 5 min incubation. Newly synthesized (labeled) mRNA was separated from pre-existing pools using thiol-specific biotinylation and fractionation. Fractionated mRNA can be used for first strand synthesis and qRT-PCR can provide estimates of de novo transcription rates for specific genes. The method can be scaled up for high throughput RNA sequencing to examine global transcription and degradation rates in response to environmental stimuli. Thiol-labeled RNA can be used to enhance RNA:protein crosslinking allowing for the characterization the mRNA-bound proteome and to identify nucleotides in protein-RNA binding sites. Examples of these applications using T. pseudonana will be presented.
69. THE DRAFT GENOME OF THE COSMOPOLITAN DIATOM CHAETOCEROS DEBILIS
Mariam R. Rizkallah1, Ahmed Moustafa2, Stephan Frickenhaus1,3, Bánk Beszteri1, Sára Beszteri1
1 Alfred Wegener Institute for Polar and Marine Research Bremerhaven, Bremerhaven, Germany
2 Department of Biology and Biotechnology Graduate Program, American University in Cairo, New Cairo, Egypt
3 Hochschule Bremerhaven, Biotechnology, Bremerhaven, Germany
The cosmopolitan diatom species, Chaetoceros debilis, has been reported to show an intense immediate response to iron fertilization in both the Southern and the subarctic Pacific oceans. Unraveling its metabolic potential at a molecular level is crucial for our understanding of marine diatoms evolution and adaptation. Here we present the findings of the ongoing C. debilis genome-sequencing project. We present the draft genome architecture and the assembly results produced by two De-Bruijn-graph based assemblers, ABySS and CLC Genomics Workbench, of 64,491,235 paired-end and 109,466,420 mate-pair Illumina reads of C. debilis genome. We also present the preliminary findings of the evolutionary analysis of potential iron storage and utilization pathways in centric diatoms. C. debilis genome will be an addition to the sequenced fraction of heterokonts in the Tree of Life, which will deepen our understanding of secondary endosymbiotic events. We are improving the genome annotation by the incorporation of transcriptomic data of another ongoing study investigating the behavior of C. debilis under iron/silica/light co-limitations. Presenting our early findings at a highly specialized conference such as yours will benefit our study through the input of the diatom scientific community.
73. TALEN-MEDIATED KNOCKOUT OF AUREOCHROME 1A IN THE DIATOM PHAEODACTYLUM TRICORNUTUM
Serif, M., Weißert, K., Rio Bartulos, C., Lepetit, B., Kroth, P.G.
Plant Ecophysiology, University of Konstanz, Universitätsstr. 10, 78464 Konstanz, Germany
One of the most important features of a model organism is the availability of an efficient reverse genetics approach, greatly facilitating characterization of gene function in vivo. Until recently, only RNAi-induced knockdown of genes was established in the diatom Phaeodactylum tricornutum. However, this procedure may result in reverting cell lines over time. In other eukaryotes, two techniques were recently established, which induce targeted DNA double-strand breaks to knock out genes irreversibly: The TALEN (Transcription activator-like effector nucleases) system and the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) system. Aureochromes are a novel type of blue-light photoreceptors discovered in 2007, which have so far only been found in Stramenopiles. In addition to a blue-light sensing LOV-domain they have a DNA-binding bZIP domain typically found in transcription factors. We used a TALEN-based approach to knock out Aureochrome 1a, one of four homologs found in P. tricornutum. The knockout strains showed a high-light acclimation phenotype similar to the previously studied knockdown strains, including significantly reduced Chlorophyll a content per cell. Additionally, we determined both the efficiency of our TALEN system, as well as the potential heterogeneity of the obtained cell lines via allele-specific PCR and genomic Southern Blot.
75. BIODIVERSITY OF EUKARYOTIC SEA ICE DIATOMS OF THE CENTRAL ARCTIC OCEAN
Anique Stecher1, 2, Stefan Neuhaus2, Benjamin Lange2, 3, Stephan Frickenhaus2, 4, Bánk Beszteri2, Peter G. Kroth1 and Klaus Valentin2
1 Department of Biology, University of Konstanz, Konstanz, Germany
2 Alfred Wegener Institute Helmholtz Center for Polar and Marine Research, Bremerhaven, Germany
3 University of Hamburg, Zoological Institute and Zoological Museum, Biocenter Grindel, Hamburg, Germany
4 Hochschule Bremerhaven, Bremerhaven, Germany
Sea ice is a large and diverse ecosystem inhabited by bacteria and protists contributing significantly to primary production in ice-covered regions. In the Arctic Ocean, sea ice consists of mixed multi-year ice (MYI) and thinner first-year ice (FYI). Due to global warming we experience a shift from MYI towards FYI. Despite the great importance of the sea ice ecosystem, little is known about its functional biodiversity, i.e. which species are actively contributing with which functions to the community. We investigated the eukaryotic biodiversity in MYI and FYI from the central Arctic Ocean using 18S rRNA and rDNA amplicons and compared the “total” biodiversity (rDNA-based) with the “active” biodiversity (rRNA-based). Groups like Ciliophora, Bicosoecida and Bacillariophyceae were over-represented in the active part of the community and grazers appear most active in one FYI station due to the advanced stage of melt compared to the other stations. Furthermore, preliminary results of transcriptomic stress experiments with an abundant naviculoid sea ice diatom show that based on physiological parameters this diatom has a broad thermal range (5 °C to -5 °C) but significantly changes its gene expression pattern at higher temperatures.
76. SKELETONEMA MARINOI AND ASSOCIATED MICROBIOME
Sylvie VM Tesson1, Alvar Almstedt1, Mats Töpel1,2, Olga Kourtchenko1, Susanna Gross1, Kerstin Johannesson1, Anders Blomberg1, Anna Godhe1
1 Department of Marine Sciences, University of Gothenburg, Sweden
2 Bioinformatics Infrastructure for Life Sciences, BILS, www.bils.se
In the context of the investigation of Skeletonema marinoi genome, a centric diatom widely distributed in Scandinavian waters, bacterial reads were unveiled, the genomes of several bacteria assembled and their relationship with S. marinoi investigated. Bacterial reads were identified in Illumina libraries sequenced from axenic cultures and in 22 SMRT cells of long pacbio reads sequenced from axenic and non-axenic cultures. In Illumina libraries, several contigs of bacterial origin grouped into islands of specific coverage and GC content. Four bacterial genomes, ranging between 2.0 to 3.8 Mb size, were assembled using long reads and SMRT technology. Taxonomy of bacterial sequences was evaluated in axenic and non-axenic cultures of S. marinoi and the location of these sequences assessed in relation to the phytoplankton cell using bioinformatic tools and in vitro cell hybridization.
79. THE SKELETONEMA MARINOI GENOME PROJECT
Mats Töpel1,2, Sylvie VM Tesson2, Magnus Alm Rosenblad2, Tomas Larsson2, Susanna Gross2, Alvar Almstedt2, Kerstin Johannesson2, Anders Blomberg2, Anna Godhe2
1 Bioinformatics Infrastructure for Life Sciences, BILS (http://bils.se/), Sweden
2 Department of Marine Sciences, University of Gothenburg, Sweden
We have sequenced the genome and transcriptome of the centric diatom Skeletonema marinoi GUMACC st54, as part of the Linnaeus Centre for Marine Evolutionary Biology (CeMEB, http://cemeb.science.gu.se/). This work is part of the Infrastructure for Marine Genetic Model Organisms (IMAGO), aimed at developing new marine model systems and provide genomic tools to study costal marine ecosystems. Skeletonema marinoi is an abundant primary producer during spring blooms in the North Atlantic and a valuable food source for zooplankton. Its generation time is 24 hours, which makes it ideal for studies of phenotypic response to environmental cues. Benthic cells act as resting stages, with up to 50 000 cells per gram of sediment, and can survive for at least hundred years and thereby provide short‐term evolutionary genomic archives in the sediment (see also the abstracts from Godhe et al. and Tesson et al.). In this project we have used Illumina paired‐end libraries of insert sizes 180, 300 and 650 bp, mate pair libraries of insert sizes 3Kb and 6Kb, together with SMRT technology long reads from Pacific Biosciences to assemble a first version draft genome, to be used for population genomics studies.
80. COMPARATIVE ANALYSIS OF CENTRIC DIATOM GENOMES OFFERS INSIGHTS INTO GENOME ARCHITECTURE AND CARBON METABOLISM
Jesse Traller1, David Lopez2, Shawn Cokus2, Olga Gaidarenko1, Orna Cook1, Aubrey Davis1, Sean Gallaher3, Marco Morselli2, Artur Jaroszewicz2, Sabeeha Merchant3, Matteo Pellegrini2, Mark Hildebrand1
1Marine Biology Research Division, Scripps Institution of Oceanography, UC San Diego, La Jolla, CA USA
2Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA, USA
3Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA USA
In the 1980’s a US government initiative to develop algal biofuels into a commercial fuel source selected Cyclotella cryptica, a coastal marine diatom, as a strain of microalgae suitable for large-scale production. Today, with the advancements in high throughput sequencing, it has become feasible to obtain high quality, cost-efficient genomic data of biofuel production strains in order to better control the biological processes affecting industrial efficiency. With that rationale, we sequenced the genome, methylome, and transcriptome of C. cryptica and used comparative genomics to elucidate carbon metabolism and gene regulation in the cell. The large (180Mbp), repeat-rich genome and unique methylation signatures in C. cryptica, especially in comparison to the closely related, highly similar, T. pseudonana, inspired compelling hypotheses regarding diatom genome scaling and cellular strategies to combat transposable element expansion. We also focused our efforts in detailed annotation of genes involved in the metabolism of carbon-rich sinks, such as triacylglycerol, chrysolaminarin, and chitin. Results from this study highlight genes, genomic and epigenetic features, and metabolic processes that are of interest from both an algal biofuel and cellular biology perspective.
83.CHARACTERISATION AND OPTIMISATION OF MIXOTROPHIC GROWTH IN PHAEODACTYLUM TRICORNUTUM
Valeria Villanova1,2, Antonio Emidio Fortunato3, Angela Falciatore, Adeline Le Monnier2, Julien Pagliardini2, Giovanni Finazzi3, Dimitris Petroutsos1
1 iRTSV, Laboratoire de Physiologie Cellulaire Grenoble, France
2 Fermentalg, Libourne, France
3 Univeristé Pierre et Marie Curie, Paris, France
Diatoms are unicellular eukaryotes that use light via photosynthesis (photoautotrophy) and carbon via respiration (heterotrophy) as energy sources for growth. The model diatom Phaeodactylum tricornutum is a potential source of triacylglycerols, used in the production of biofuel. However, this diatom does not process the carbon in the dark and the cost of photoautotrophic growth makes it an expensive source of these molecules. The simultaneous use of photosynthesis and respiration (mixotrophy) in P. tricornutum can increase the biomass productivity and reduce the energy cost for its industrial exploitation. Here, the mixotrophic growth of P. tricornutum using glycerol as carbon source has been tested. The addition of this compound to the optimised growth medium enhances biomass production and triacylglycerol accumulation by a factor of two. Optimization has been assessed measuring biomass production (through measurements of growth rate), photosynthetic activity (fluorescence imaging and absorption spectroscopy) and lipid content. Moreover, transcriptomic analysis comparing wild type cells (grown with and without glycerol) to evaluate changes in gene expression has been performed. By combining this approach to genome based analysis, we pinpoint putative candidates for metabolic engineering. The optimized condition and the interesting mutant lines generated will then scale-up in 2 liters photobioreactors.
89. TEMPO AND MODE OF DIATOM PLASTID GENOME EVOLUTION
Mengjie Yu1, Matt P Ashworth1, Edward C Theriot2, Jamal S Sabir3, Robert K Jansen1,3
1 Department of Integrative Biology, University of Texas at Austin, Austin TX 78712, USA
2 Texas Memorial Museum, University of Texas at Austin, Austin, 78712, USA
3 Biological Sciences Department, Faculty of Science, King Abdulaziz University, Saudi Arabia
Knowledge of genome architecture evolution and nucleotide substitution rates are essential for our understanding of the tempo and mode of molecular sequence evolution. So far, fifteen diatom plastid genomes have been published, and they show variable genome sizes and extensive genome rearrangements. Our recently published research added another seven diatom plastid genomes, revealing conserved gene order and expanded inverted repeats in plastid genomes of the order Thalassiosirales and documenting the first loss of photosynthetic genes in a photoautotrophic diatom (Rhizoselenia imbricata). Our ten newly finished plastid genomes (Proboscia, Guinardia striata, Actinocyclus sultilus, Rhizosolenia setigera, Eunnotogramma, Plagiogrammopsis, Attheya, Plagiogramma, Pteroncola, Licmorphora ) show extensive genome rearrangements with genome size ranging from 121,011bp to 201,816bp. We have an additional 16 plastid genome sequences currently in progress. We will be presenting diatom phylogeny using the 48 complete plastid genomes, and test the efficacy of genome rearrangement data in resolving phylogenetic relationships. We will also be presenting the first genome-wide evolutionary rate analysis in diatoms, and implications of rate variation for understanding diatom evolution.
5. GLOBAL PATTERNS OF DIATOM DIVERSITY DERIVED FROM THE TARA OCEANS EXPEDITION
Chris Bowler, Shruti Malviya, Flora Vincent, Lucie Bittner, Gipsi Lima-Mendez, Stéphane Audic, Sebastien Colin, Nicolas Henry, Eleonora Scalco, Adriana Zingone, Colomban de Vargas, Jeroen Raes, and the Tara Oceans Consortium
Ecology and Evolutionary Biology Section, Institut de Biologie de l’Ecole Normale Supérieure (IBENS), Paris, France
Diatoms are believed to be the most important group of photosynthetic eukaryotes in the oceans. Understanding patterns of diatom distribution is therefore important for understanding marine ecosystem functioning. From the Tara Oceans global sampling expedition a total of ca. 14.5 million V9-rRNA ribotypes assigned to diatoms derived from 47 sampling stations that span the global ocean euphotic zone are being analyzed to understand diatom community composition. The dataset contains 65,000 unique ribotypes, present in a wide range of abundances, and represents approximately 3,300 Operational Taxonomic Units. Some of these localize to specific areas such as the Southern Ocean or to sites of diatom blooms, or display interesting biogeographical patterns such as commonalities along the Agulhas current originating from the southwest Indian Ocean. The data also reveal an unexpectedly high level of diatom diversity in the open ocean. Clustering with environmental parameters further reveals correlations of some diatom groups with net primary production and carbon flux. Global comparisons of diatom co-occurrence and exclusion patterns with ribotypes from other organisms provide insights into the interactions of diatoms with other planktonic organisms.
16. CHARACTERIZING SOUTHERN OCEAN DIATOM COMMUNITY COMPOSITION TO ESTABLISH ECOLOGICALLY RELEVANT CULTURE REPRESENTATIVES FOR IRON PHYSIOLOGY EXPERIMENTS
Laura Z. Filliger1, Tom O. Delmont3, Anton Post2,3, Bethany D. Jenkins1,2
1Department of Cell and Molecular Biology, 2Graduate School of Oceanography, University of Rhode Island, Kingston, RI, USA,
3Marine Biology Laboratory, Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Woods Hole, MA, USA
Southern Ocean (SO) diatoms are predicted to be growth limited for iron, an essential protein cofactor. We are particularly interested in delineating the iron stress response in SO diatoms as they are the base of the SO food web. To develop ecologically relevant laboratory models, we established a culture collection of >300 isolates from two austral summer and winter cruises conducted in 2013-2014. To identify SO species with broad iron tolerance, we characterized in situ diatom community composition across regions of varying iron levels. This was accomplished via high-throughput sequencing of the variable V4 region of the 18S rDNA amplified with diatom-targeted primers. Fragilariopsis sp. and Pseudo-nitzschia sp. dominated most of the sampled areas. We are currently determining the phylogenetic resolution we achieve with the V4 region by comparing isolate and community amplicon data. As our culture collection contains representatives of the major taxa identified with molecular barcoding, we will be able to conduct iron physiology experiments with relevant culture representatives. This will allow us to probe the physiological status of diatoms important in different regions of the SO, and to determine if iron limitation is experienced more or less severely in different diatoms.
26. USING TARGETED METABOLOMICS TO ASSESS COBALAMIN DEMAND IN DIATOM CULTURES AND SURFACE OCEAN
Katherine Heal1, Willow Coyote-Maestes1, E. Virginia Armbrust1, Dave Stahl1, Al Devol1, Jim Moffett2, Anitra Ingalls1
1 University of Washington, Seattle, WA, USA
2 University of Southern California, CA, USA
Investigations into the role of cobalamin (Vitamin B12) in diatom-dominated ecosystems have found this important micronutrient can co-limit primary productivity, control phytoplankton biodiversity, and have implications on the efficiency of the metabolism of these globally important organisms (reviewed in Bertrand et al 2012). Despite the growing interest in this cobalt-containing compound, direct measurements of cobalamin in seawater, either dissolved or particulate are few and far between (Sañudo-Wilhelmy et al 2014). Using targeted metabolomics (Heal et al 2014), we compare active and inactive forms of cellular and dissolved cobalamin in diatom cultures to elucidate how metabolite pools reflect cobalamin demand in simple systems. We then use this same technique along a transect through coastal waters and into the high nutrient low chlorophyll gyre in the North Pacific as evidence for cobalamin demand in diatom dominated surface waters.
62. DIATOMS: THE ROLE OF PROGRAMMED CELL DEATH IN CARBON SEQUESTRATION IN THE ROSS SEA
Mónica V. Orellana1&2, Allison Lee2, Adrian Lopez Garcia de Lomana2, Meredith Jennings3, Sarah Bercovici3, Nitin S. Baliga2 and Dennis A. Hansell3
1 Institute for Systems Biology and University of Washington, Seattle, WA, USA
2 Institute for Systems Biology, Seattle, WA, USA
3 University of Miami, Rosenstiel School of Marine and Atmospheric Science, FL, USA
The Southern Ocean plays a critical role for carbon sequestration in the oceans. The flux of carbon (C) and nitrogen (N) export to the deep ocean depends on the efficiency of the biological pump transporting biomass to depth. This study focuses on the impact of programmed cell death (PCD) in the Ross Sea, observed in the late austral summer during TRACERS (2013). Program cell death modulates the turnover of phytoplankton in the oceans, however very few field data exists recapitulating this phenomenon. We present evidence of diatoms undergoing PCD and their proximate causes in the Ross Sea, as well as examine other primary groups of organisms undergoing PCD in the water column. Flow cytometric and phylogenetic analyses of the populations undergoing PCD indicate high occurrences of diatoms within the eukaryote community at the ocean surface, while prokaryotes undergoing PCD were found at depth. In addition, we discuss investigations conducted to determine the role of PCD in mediating carbon export through the production of biopolymers as free and assembled polymer gels.
17. REVEALING THE STRUCTURAL BASES FOR LIGHT UTILIZATION IN DIATOMS
Serena Flori1, Benoit Gallet2, Christine Moriscot2, Simona Eicke3, Samuel Zeeman3, Norbert Rolland1, Marianne Tardif4, Myriam Ferro4, Guy Schoehn2, Dimitris Petroutsos1, Denis Falconet1 and Giovanni Finazzi1.
1 Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'Energie Atomique (CEA)-Université Grenoble Alpes, INRA, institut de Recherche en Sciences et Technologies pour le Vivant (iRTSV), CEA Grenoble, 38054, Grenoble, France
2 UMR5075, Institut de Biologie Structurale (IBS ), Grenoble, France.
3 Dpt of Biology, ETH Zurich, Switzerland.
4 EDyP, iRTSV, CEA Grenoble.France.
Over the past ~ 35 million years, diatoms have been one of the most successful classes of photosynthetic marine eukaryotes, largely contributing to the biological carbon pump. Today their photosynthetic activity is probably equivalent to that of terrestrial rainforests. Photosynthesis in diatoms occur within a peculiar chloroplast originated by a secondary endosymbiosis event. Therefore, no differentiation of the photosynthetic membranes between appressed regions (Grana= rich in photosystem II, PSII) and non-appressed regions (Stroma lamellae= rich in photosystem I, PSI) is seen, unlike plants. This raises the question of optimum photosynthetic light harvesting and electron flow, because absence of spatial segregation of the PSs might trigger energy waste via direct transfer from PSII to PSI (spillover). This hypothesis was challenged in Phaeodactylum tricornutum measuring energy transfer to P700 (the primary electron donor to PSI) in the absence and presence of DCMU treated cells (to block light utilisation by PSII). No differences were found, showing that light absorbed by PSII is not shared with PSI (i.e. no spillover occurs). This suggests that no physical contact exists between the photosystems. Biochemical and immunolocalization analysis confirmed this conclusion, revealing a refined compartmentation of photosystems between the external and the innermost thylakoid membranes. We propose that this particular spatial arrangement of photosynthetic complexes allows optimum partitioning of absorbed light between the photosystems, while not restraining electron flow capacity, as required for optimum photosynthesis.
22. SEPARATED EFFECTS OF PH AND PCO2 ON PHOTOSYNTHESIS AND RESPIRATION IN THALASSIOSIRA WEISSFLOGII
Johanna A.L Goldman, Francois M.M. Morel
Department of Geosciences, Princeton University, Princeton, NJ 08544, USA
The rise of atmospheric CO2 leads simultaneously to an increase in CO2 concentration and a decrease of pH in the ocean. The potential effect of these changes on diatoms is critical since they produce ~40% of global primary production. Previous workers have obtained varying results, sometimes contradictory, on the simultaneous effect of increasing pCO2/decreasing pH on the net photosynthesis of diatoms. This study investigates the separated effects of pCO2 and pH on gross photosynthesis and respiration at different light intensities in the centric diatom Thalassiosira weissflogii. We used Membrane Inlet Mass Spectrometry, with H218O as a tracer, to follow the changes of 18O16O (gross photosynthesis) and 16O2 (net photosynthesis) at different light intensities. We observed little to no effect of either pH or pCO2 on either net or gross photosynthesis at any light intensity. We also observed decreasing respiration in the light with increasing light intensity. These results are surprising considering the known down-regulation of this diatom carbon concentrating mechanism (CCM) at increasing pCO2, which should lead to a decrease in the energetic cost of carbon fixation. On-going experiments are examining why changes in CCM activity under different pH/pCO2 conditions are not affecting gross and net photosynthesis in this organism.
25. INVESTIGATING CYCLIC AMP AS A MEDIATOR OF CO2-SENSING IN THE DIATOM THALASSIOSIRA PSEUDONANA
Ryan D. Groussman, Gwenn M. M. Hennon, E. V. Armbrust
School of Oceanography, University of Washington, USA
Anthropogenic emissions are projected to double atmospheric concentrations of CO2 by the centuries’ end and acidify the oceans, fundamentally changing the marine environment. Since diatoms compose 40% of marine primary production, it is important to understand their response to increasing CO2. In previous work, we identified genes and gene clusters with differential expression under elevated CO2 in the model diatom Thalassiosira pseudonana. One cluster of genes that were down-regulated under elevated CO2 encode putative carbon concentrating mechanism (CCM) genes including carbonic anhydrase, delta-CA3. These genes share an upstream cis-regulatory motif characterized to be involved in the regulation of CCM genes in Phaeodactylum tricornutum, a distantly related diatom, with cyclic AMP (cAMP) as the second messenger. It is unknown whether cAMP plays a similar role in CO2-responsive gene regulation in T. pseudonana. To address this, we grew T. pseudonana under high and low pCO2 conditions and sampled cultures prior to or following exposure to 3-isobutyl-1-methylxanthine (IBMX), which raises intracellular cAMP concentrations. After IBMX treatment, we observed a significant decrease in delta-CA3 transcript abundance. These results support the role of cAMP as a key messenger in CO2-sensing and acclimation, allowing new mechanistic insights into diatom responses to changing ocean environments.
30. CONTRASTING PHYSIOLOGICAL AND BIOCHEMICAL ADAPTATIONS OF TWO STRAINS OF THE MODEL DIATOM THALASSIOSIRA OCEANICA TO FE AND/OR CU LIMITATION
A. Hippmann1, M. Alami2, A. Allen3, J. McCrow3, L. Foster4, B. Green2, M. Maldonado1
1 University of British Columbia, Department of Earth Ocean and Atmospheric Science, Earth Sciences Building, 2207 Main Mall, Vancouver, V6T 1Z3, Canada, email@example.com, +1-604-827-5459
2 University of British Columbia, Department of Botany, Room #3529 - 6270 University Boulevard, Vancouver, B.C., V6T 1Z4, Canada
3 J. Craig Venter Institute, San Diego, California 92121, USA
4 Centre of High-Throughput Biology, 2125 East Mall, Vancouver, BC V6T 1Z4, Canada
Iron plays a significant role in controlling marine primary productivity. Approximately 30% of the global ocean is iron limited. In these regions, free iron concentrations are so low that it has been questioned how iron dependent phytoplankton are able to survive in such Fe-deficient environments. Thalassiosira oceanica has been used as a model diatom to elucidate their physiological adaptations to these low iron conditions. Previous studies have shown that T. oceanica has lowered its iron requirements and increased the efficiency of its iron acquisition mechanisms. Both strategies require a higher physiological copper dependency. However, the underlying biochemical adaptations of this organism remain unknown. Recently, the genome, as well as the first proteomic and transcriptomic analysis of T. oceanica grown under different Fe levels, was published. To further our understanding of the interplay between iron and copper physiology of open ocean diatoms, we examined an array of physiological responses to varying degrees of iron, copper and iron/copper co-limitation. To investigate the underlying biochemistry, we employed differential proteomic analysis using stable isotope labeling and LC-MS/MS.
This work was supported by the Natural Sciences and Engineering Council of Canada.
32. KNOCKDOWN OF PHOSPHOENOLPYRUVATE CARBOXYKINASE INCREASES TRIACYLGLYCEROL ACCUMULATION WITHOUT COMPROMISING GROWTH IN PHAEODACTYLUM TRICORNUTUM
Hanhua Hu, Yang Juan
Diatom Biology Group, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
The tricarboxylic acid (TCA) cycle is a key metabolic pathway that unifies carbohydrate, lipid and protein metabolism. In Phaeodactylum tricornutum, accumulation of triacylglycerols (TAGs) under nitrogen stress is a consequence of allocation of carbon mainly from the intermediates of TCA cycle. A mitochondrial-localized phosphoenolpyruvate carboxykinase (PEPCK), which converts TCA intermediate oxaloacetate (OAA) into phosphoenolpyruvate and CO2, was significantly upregulated at both the mRNA and protein levels during TAG accumulation in P. tricornutum. Knockdown of pepck would decrease the carbon loss resulting from decarboxylation of OAA. Both PEPCK transcript abundance and enzyme activity were reduced significantly in two RNA interference lines compared with wild-type cells. Although the two pepck knockdown lines exhibited slower growth and lower photosynthetic activity during cellular exponential growth, their cell density was almost the same with that of wild-type at the stationary growth phase. TAG content per cell at day 10 in the pepck mutants increased by 15~21% compared with what was observed in wild-type cells. The neutral lipid content was increased by 76~85% via nitrogen-deprivation treatment for 48h. This study, for the first time, provided a new strategy, which succeeded in increasing lipid accumulation in diatoms by decreasing the loss of carbon skeletons without compromising growth.
35. ACCLIMATION STRATEGY TO LIGHT/SHADE ENVIRONMENTS IN DIATOM
Natsuko Inoue-Kashino1, Shimpei Aikawa1, Kumiko Fujimoto1, Tomoko Ishihara1, Kentaro Ifuku2, Sakae Kudoh3,4, Kazuhiko Satoh1, Yuichiro Takahashi5 and Yasuhiro Kashino1
1Department of Life Science, University of Hyogo, 3-2-1 Kohto, Kamigohri, Ako-gun, Hyogo 678-1297, Japan
2Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
3National Institute of Polar Research, 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan
4Department of Polar Science, The Graduate University for Advanced Studies, 10-3 Midori-cho, Tachikawa, Tokyo 190-8518, Japan
5Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
Marine diatoms contribute greatly to the global photosynthetic crop. They perform photosynthesis by acclimating to the wide range of ambient light intensities in the ocean. Regulation of photosynthetic electron transport-related system in a marine pennate diatom Phaeodactylum tricornutum and a marine centric diatom Chaetoceros gracilis, grown under wide range of irradiances were assessed. The ratio of accessary pigments to chlorophyll a was not changed remarkably but that of xanthophyll pigment increased under high irradiance. Delivery of the captured light energy to the reaction centers changed drastically depending on the growth irradiance. The changing manner was different between the two diatoms; more light energy to photosystem I under dim light in P. tricornutum and opposite in C. gracilis. The chlorophyll a-specific amount of the reaction centers was kept almost the same irrespective of the growth irradiance in both diatoms. Therefore, light-harvesting complex shuttles between the two reaction centers depending on the growth irradiance. However, the mechanism is different from the state transition in other photosynthetic organisms in terms of scale and duration, namely state conversion. The dynamic state conversion in diatoms should achieve effective photosynthesis under the wide range of light intensities in the ocean.
37. FUNCTIONAL STRUCTURE OF LIGHT HARVESTING PROTEIN COMPLEX IN CHAETOCEROS GRACILIS
Tomoko Ishihara1, Natsuko Inoue-Kashino1, Kentaro Ifuku2, Eiki Yamashita3, Yuko Fukunaga1, Yuri Nishino1, Atsuo Miyazawa1, Yasuhiro Kashino1
1Graduate School of Life Science, University of Hyogo
2Graduate School of Biostudies, Kyoto University
3Institute for Protein Research, Osaka University
Diatom captures light energy using an antenna system called fucoxanthin-chlorophyll a/c binding protein complex (FCP). Diatom genome contains about 30 genes for FCP-proteins. However, functional difference among those FCP-proteins is not known. Toward the understanding of their specific function, we analyzed the structural linkage between them and that with reaction center complexes. FCP complex was purified from a marine centric diatom, Chaetoceros gracilis by sucrose density gradient centrifugation after solubilization by a detergent. Measurement of dynamic light scattering indicated that the purified FCP complex was in only single oligomerization state. The amount of Chl a bound to the purified FCP complex was estimated to be >60% of total Chl a in solubilized thylakoid. Nonetheless, the complex was composed of only three distinguished polypeptides among nearly 30 FCP-proteins. Major two proteins were identified as Fcp3- and Fcp4-equivalent proteins by internal amino acid sequencing and agarose-based isoelectric two-dimensional electrophoresis. Fluorescence induction and emission spectra showed that the main function of this complex is the light capturing rather than non-photochemical quenching. This FCP complex that carries abundant pigments should play an important role for diatoms to acclimate to the changing environment to achieve efficient photosynthesis.
38. DISSECTING THE MOLECULAR BASES OF DIATOM PHOTOPROTECTION
Lucilla Taddei1, Giulio R. Stella1, Alessandra Rogato1,2, Dimitris Petroutsos3, Giovanni Finazzi3, Marianne Jaubert1, Angela Falciatore1
1 Laboratory of Computational and Quantitative Biology, UMR7238 UPMC/CNRS, Paris, France
2 Institute of Biosciences and Bioresources, CNR, Naples, Italy
3 Laboratoire de Physiologie Cellulaire et Végétale, UMR5168 CNRS/UJF/INRA/CEA Grenoble, France
Diatoms show optimal photosynthetic activity over a wide range of environments. For example, they show an outstanding capacity to cope with light stress and dissipate excess energy in the nonphotochemical quenching (NPQ) photoprotecting process. We notably uncovered the key function played by a member of the light-harvesting complex stress-related family protein, named LHCX1, in the regulation of NPQ and growth. Moreover, we recently observed that lhcx-1 knock-down cells display a reduced content of some photosynthetic proteins but recover a normal NPQ capacity under prolonged high light treatment. Reduced respiration capacity is also observed in these cells, suggesting the existence of a cross-talk between chloroplast and mitochondrion regulation. In parallel, we are also characterizing the multiple genes encoding the putative enzymes of the xanthophyll cycle, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP)-like proteins. Preliminary characterization of knock-down lines for VDR and VDL2 showed a reduction of long-term photoprotection ability in high light conditions, as oppose to the phenotype observed for lhcx1 KD lines. These data strongly support the existence of a tightly interconnected network of chloroplast regulators controlling diatom acclimation capacity.
40. UNIQUE KINETIC PROCESS OF TRIGLYCERIDE PRODUCTION IN A MARINE CENTRIC DIATOM, CHAETOCEROS GRACILIS
Yasuhiro Kashino, Ryoya Takeda, Yuko Fukunaga, Yuri Nishino and Atsuo Miyazawa
Graduate School of Life Science, University of Hyogo, 3-2-1 Kohto, Kamigohri, Ako-gun, Hyogo 678-1297, Japan
Diatom is one of the important candidate organisms for a platform of renewable and sustainable energy production since some diatoms accumulate high amount of triglyceride (triacylglyceride; TAG) as oil body inside the cell. Characteristics of the growth and TAG accumulation were assessed in a marine centric diatom, Chaetoceros gracilis by comparing a marine pennate diatom Phaeodactylum tricornutum. In C. gracilis, TAG accumulation began at an early stage of exponential phase although in P. tricornutum, TAG was poor during the exponential phase to be increased in the stationary phase. Therefore, the property of TAG accumulation was different from other well-characterized microalgae including P. tricornutum in which nitrogen deprivation that accompanies suppression of growth induces TAG accumulation. The changes in fatty acid composition during the growth suggested that fatty acid components in TAG are synthesized de novo in the period of exponential phase. The regulation mechanisms of fatty acid and TAG metabolism seem to be unique in C. gracilis.
41. INTRASPECIES HOST SPECIFICITY OF DNA/RNA VIRUSES THAT INFECT MARINE PLANKTONIC DIATOM CHAETOCEROS TENUISSIMUS
Kei Kimura1 & Yuji Tomaru2
1 Institute of Lowland and Marine Research, Saga University, Japan
2 National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Japan
Until today, two groups of diatom viruses have been recognized; single-stranded DNA virus (ssDNAV) and single-stranded RNA virus (ssRNAV). Among these host-virus systems, the marine planktonic diatom Chaetoceros tenuissimus is the sole host species that has both DNA and RNA viruses. We conducted a cross reactivity test between 108 C. tenuissimus strains and 233 viral isolates (83 ssRNAV and 150 ssDNAV isolates). In terms of host strain specificities, the ssRNAV isolates were separated into two groups; one is narrow host rages and the other is wide. The host strain specificities of the ssDNAVs were diverse and divided into 6 groups. The partial viral genome sequencing analysis showed no evidence for the relationship between the infection specificities of the ssRNAVs and their sequences, including replication related and capsid protein genes, and untranslated region. The viral capsid protein gene of ssDNAVs, while, possibly relates to their specificities. Adsorptions of ssDNAVs to the host cells might be an important step for their specific infection. The further sequencings of the viral genome should be necessary to detect the core region to determine the host specificities of diatom viruses.
53. DEUTERIUM DISCRIMINATION IN LIPIDS VARIES INVERSELY WITH SALINITY IN CONTINUOUS CULTURES OF THE DIATOM THALASSIOSIRA PSEUDONANA
Ashley E. Maloney, Avery L.C. Shinneman^, Kathleen Hemeon, Julian P. Sachs
University of Washington, School of Oceanography, College of the Environment, Box 355351, Seattle, WA 98195, USA
^Present address: University of Washington at Bothell, School of Interdisciplinary Arts and Sciences, Box 358500, 18115 Campus Way NE Bothell, WA 98011, USA, firstname.lastname@example.org
The hydrogen isotope ratio (2H/1H or D/H, reported as D) of lipids in microalgae is typically 100-400‰ lower than extracellular water. This is attributed to the very low D value of hydride derived from photosynthetically produced NADPH. Recently it has been shown that D/H fractionation in lipids decreases as salinity increases, attracting much attention due to potential application of Dlipid as a paleosalinity proxy. However the mechanism responsible for this phenomenon remains uncertain, chiefly because no study has been conducted with continuous cultures. We report the lipid D/H fractionation response to salinities between 9-40 ppt in steady state continuous cultures of Thalassiosira pseudonana. Fatty acids and 24-methyl-cholesta-5,24(28)-dien-3β-ol from six continuous cultures decreased linearly as salinity increased by 1-1.3‰ ppt-1. As growth rate remained constant, it did not contribute to variable fractionation as has been previously hypothesized. We propose that increased exchange of hydrogen between intracellular water and organic matter, and/or increased metabolic production of D-enriched NADPH are potential mechanisms responsible for the observed D-enrichment of lipids at high salinity. A simple steady-state box model of hydrogen cycling in a population of phytoplankton was designed to test if lipid D/H is sensitive to these cellular processes.
55. PEERING INTO A ‘BLACK BOX’ MEASUREMENT – RESOLVING GROUP-SPECIFIC SILICON UPTAKE RATES IN A NATURAL DIATOM ASSEMBLAGE
Heather McNair1, Mark Brzezinski1, 2, Jeffrey Krause2, 3, 4
Field experiments to determine the concentration dependence of silicic acid uptake provide information regarding the degree to which silicic acid limits the rate of biogenic silica production in the surface ocean. Such data are vital for identifying conditions that restrict diatom production. However, their relevance to diatom population ecology is limited as the measurement averages the responses of individual diatom species. Resolving taxon-specific responses would reveal physiological differences among diatom taxa that may contribute to species succession driven by competition for silicic acid. We investigated the silicic acid uptake kinetics for a natural diatom assemblage off the California coast to illustrate the utility of a new quantitative PDMPO labeling method for assessing silica production by individual diatom cells. Eleven genera were present, but Chaetoceros spp. accounted for 93% of cells. The half-saturation constant and maximum uptake values for the entire assemblage determined using 32Si were 2.59 µM and 0.012 h-1, respectively and uptake was limited to 15% of maximum at the ambient silicic acid concentration. The half saturation constants and maximum Si uptake rates for individual species, and their contribution to total-assemblage biogenic silica production will be discussed, providing an unprecedented view of functional variability among diatom in natural assemblages.
65. MEMBRANE GLYCEROLIPID REMODELING TRIGGERED BY NITROGEN AND PHOSPHORUS STARVATION IN PHAEODACTYLUM TRICORNUTUM
Heni Abida1, Lina-Juana Dolch2, Coline Meï2, Valeria Villanova3, Melissa Conte2, Maryse A. Block2, Giovanni Finazzi2, Olivier Bastien2, Leïla Tirichine1, Chris Bowler1, Fabrice Rébeillé2, Dimitris Petroutsos2,*, Juliette Jouhet2,*, Eric Maréchal2,*
1 Environmental and Evolutionary Genomics Section, Institut de Biologie de l'École Normale Supérieure, CNRS UMR 8197, INSERM U1024, 46 rue d'Ulm, 75005 Paris, France.
2 Laboratoire de Physiologie Cellulaire et Végétale, Unité mixte de recherche 5168 CNRS – CEA – Université Grenoble Alpes, Institut de Recherche en Sciences et Technologies pour le Vivant, CEA Grenoble, 17 rue des Martyrs, 38054, Grenoble Cedex 9, France.
3 Fermentalg SA, 4 bis rue Rivière, F-33500 Libourne, France.
Diatoms constitute a major phylum of phytoplankton biodiversity in ocean and fresh water ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane glycerolipids has not yet been studied. Our goal was first to define a reference for the glycerolipidome of the marine model diatom Phaeodacylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the glycerolipid profile of Phaeodactylum grown with various levels of nitrogen or phosphorus supplies. Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. We propose that phospholipids are secondary P-storage molecules broken down upon P deprivation, while non-phosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol.
74. INVESTIGATING LIPID PRODUCTION IN A GENOME SCALE METABOLIC MODEL OF PHAEODACTYLUM TRICORNUTUM
Dipali Singh, Mark Poolman, David Fell
Department of Biological and Medical Science, Oxford Brookes University, Oxford, UK OX3 0BP
A genome scale metabolic model (GSM) describes the metabolic interactions in an organism and allows thorough understanding of the metabolism by characterizing flux distribution in the network at various environmental and genetic conditions. The goal of this project is to investigate the metabolism of Phaeodactylum tricornutum using this approach. A GSM for Phaeodactylum tricornutum has been constructed and consists of 509 reactions excluding transporters and is fully compartmentalized (cytosol, mitochondria, chloroplast and peroxisome). It is capable of producing all major biomass components (amino acids, nucleotides bases, lipid, carbohydrate) in phototrophic and mixotrophic conditions. The model was analysed, using Flux Balance Analysis (FBA), over a range of light intensities to identify the response of reactions. This showed that flux in photorespiration increases with increasing light intensity with a concomitant excretion of glycollate. However, at higher light intensity glycollate is not excreted rather it is recycled, ultimately resulting in increased lipid production. Therefore, it is proposed that in P. tricornutum lipid production increases at high light level and might serve as photoprotective mechansim.
Funding: European Unions Seventh Framework Programme, grant agreement PITN-GA 2012-316427 http://www.accliphot.eu/
88. GROWTH AND CELL WALL MORPHOGENESIS OF TRIPARMA LAEVIS (PARMALES) UNDER SILICON-LIMITATION
Kazumasa Yamada1, Mutsuo Ichinomiya2, Akira Kuwata3, Mitsunobu Kamiya4, Kaori Ohki4, Kenji Saitoh5, Yoji, Nakamura5, Naoki Sato6, Shinya Yoshikawa4
1 Field Science Center for Northern Biosphere, Hokkaido University, Muroran, Japan
2 Faculty of Environmental & Symbiotic Sciences, Prefectural University of Kumamoto, Japan
3 Tohoku National Fisheries Research Institute, Japan
4 Faculty of Marine bioscience, Fukui Prefectural University, Japan
5 National Research Institute of Fisheries Science, Japan
6 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Japan
The order Parmales is a group of small-sized unicellular marine phytoplankton. The cell wall of Triparma laevis (Parmales) consists of 8 siliceous plates. Phylogenetic and morphological analyses suggest that Parmales is evolutionary close relationship to diatoms. We have studied the effect of silicon-limitation on growth and plate formation of T. laevis. To control the silicon concentration in the medium, we have established the batch culture of T. laevis using artificial seawater. Almost all cells had typical cell walls when they grew in the medium with sufficient silicate. However, plate formation became incomplete when cells were cultured in the low silicate medium (Ca. 10 μM of silicate). All cells finally lost all plates in the medium with lower than ca. 1 μM of silicate. However, silicon-limitation did not affect growth rate. Cells continuously grew with the same rate even after all plates were lost. When cells without plates were transferred to the medium containing 100 μM of silicate, all plates were regenerated in 36% of the cells within 24 h. These results indicate that the response to silicon-limitation of T. laevis is different from that of diatoms, the cell division of which generally arrests under such condition.
87. A REPRESENTATIVE MODEL OF THE GLIDING MOTION OF ADIATOM, BACILLARIA PARADOXA, PREDICTEDFROM THE ACTION OF ELASTIC FIBRILS
Nozomi Yamaoka1, Yasutaka Suetomo2, Tohru Yoshihisa1and Seiji Sonobe1
1 Grad. Sch. Life Sci., Univ. Hyogo, Japan
2 Iwakuni City Microlife Museum, Japan
Bacillaria paradoxa belongs to pinnate diatom and forms a colony consisting of 2-100 cells. Adjacent cells show active gliding each other, but its mechanism and physiological meanings are not understood. Alexa 488-phalloidin staining revealed that two actin bundles are present along with the raphe, elongated slit in the frustule. Both latrunculin B, an actin polymerization inhibitor, and 2, 3-butanedione monoxime, a myosin inhibitor, inhibited gliding motion, suggesting an essential role of actomyosin system.We found that a single cell separated from a colony shows a motion in back and forth at a point attaching to the substratum, and succeeded in observing a motion of plastic beads attached to the cell surface at the rapha. We believe that this motion reflect the motion of the elastic fibrils extending from raphe. Here, we present a curtain hook model for the gliding motion of Bacillaria paradoxa.
93. MOLECULAR CONTROL OF LIGHT INTENSITY ACCLIMATION IN THE MODEL DIATOM, PHAEODACTYLUM TRICORNUTUM
Ananya Agarwal 1, 2, Orly Levitan 2 , Maxim Y. Gurbanov 2 and Paul G. Falkowski 2
1Department of Microbiology & Biochemistry, Rutgers University, New Jersey, USA
2 Environmental Biophysics and Molecular Ecology Program, Dept. of Marine and Coastal Sciences, Rutgers University, New Jersey, USA
In the ocean phytoplankton experience variations in spectral irradiance from milliseconds to days. Consequently, these organisms have evolved a nested set of strategies to accommodate variations in light over these times scales. On time scales of days, many phytoplankton species can acclimate via changes in the concentration of light harvesting complexes and/or reaction centers. This reversible photoacclimation process appears to be transduced via the redox state of the plastoquinone pool by an unknown mechanism. We aim to understand the signal transduction mechanism involved in photo-acclimation in the model diatom Phaeodactylum tricornutum. Experimentally, the cells were maintained in semi-continuous exponential growth phase at 18 °C under continuous light provided from white LED’s at 20, 40, 180, 450, 715 and 940 µmol photons m-2 s-1. Growth rates (μ) varied hyperbolically as a function of incident irradiance. As predicted, cultures accumulate the most chlorophyll pigment at 20 mmol quanta m-2, and the least at 940 mmol quanta m-2 with an accompanying change in the cross-section of PSII (σPSII). The change in pigment content was associated with an optical packaging effect and strongly influenced the redox state of the plastoquinone pool at the growth irradiance of the cells.