Matilde Skogen Chauton

Gene regulation of carbon fixation, storage, and utilization in the diatom Phaeodactylum tricornutum acclimated to light/dark cycles.

Summary: Analyses of gene regulation and cell chemistry in acclimated fed batch cultures of Phaeodactylum tricornutum identify genes and metabolic pathways of carbon metabolism, and the subcellular localization and timing of genes and processes during light/dark cycles. The regulation of carbon metabolism in the diatom Phaeodactylum tricornutum at the cell, metabolite, and gene expression levels in exponential fed-batch cultures is reported. Transcriptional profiles and cell chemistry sampled simultaneously at all time points provide a comprehensive data set on carbon incorporation, fate, and regulation. An increase in Nile Red fluorescence (a proxy for cellular neutral lipids) was observed throughout the light period, and water-soluble glucans increased rapidly in the light period. A near-linear decline in both glucans and lipids was observed during the dark period, and transcription profile data indicated that this decline was associated with the onset of mitosis. More than 4,500 transcripts that were differentially regulated during the light/dark cycle are identified, many of which were associated with carbohydrate and lipid metabolism. Genes not previously described in algae and their regulation in response to light were integrated in this analysis together with proposed roles in metabolic processes. Some very fast light-responding genes in, for example, fatty acid biosynthesis were identified and allocated to biosynthetic processes. Transcripts and cell chemistry data reflect the link between light energy availability and light energy-consuming metabolic processes. Our data confirm the spatial localization of processes in carbon metabolism to either plastids or mitochondria or to glycolysis/gluconeogenesis, which are localized to the cytosol, chloroplast, and mitochondria. Localization and diel expression pattern may be of help to determine the roles of different isoenzymes and the mining of genes involved in light responses and circadian rhythms.

High-resolution magic angle spinning 1H NMR analysis of whole cells of Thalassiosira pseudonana (Bacillariophyceae): Broad range analysis of metabolic composition and nutritional value

Chemical composition of the microalga Thalassiosira pseudonana Hasle & Heimdalwas studied with different proton nuclearmagnetic resonance (1H NMR)techniques, and by comparing NMR spectrafrom extraction samples with a spectrumfrom a sample of whole cells we show thathigh-resolution magic angle spinning (HRMAS) 1H NMR can be used for broadrange analysis of metabolic composition inmicroalgal whole cells. Signals fromimportant metabolites such aspolyunsaturated fatty acids (PUFAs)eicosapentaenoic (EPA) and docosahexaenoic(DHA) acids were seen in a 1H NMRspectrum of lipophilic extract, andpossibly also signals from the carotenoidfucoxanthin. In a spectrum of hydrophilicextract we assigned signals to amino acidssuch as glutamine (Gln) and glutamic acid(Glu), carbohydrate and ATP. These findingswere compared to a spectrum of HR MAS1H NMR analysis of whole cells, whereit was possible to find signals coincidentwith the different metabolites seen inspectra of the extraction samples. Sincethe position of resonance peaks in a NMRspectrum depends on the chemicalsurroundings of each atom at the time ofanalysis some peak shift differencesbetween extract and whole cell samplespectra may occur, but signal shifts werenot significantly different between theanalyses here. In addition, application ofHR MAS highly increased spectral resolutionin the complex whole cell sample. Wetherefore suggest that HR MAS 1H NMRanalysis is a suitable analysis tool tostudy metabolic composition directly onwhole cells of microalgae, making itpossible to study a broad range ofmetabolites simultaneously without tediousextraction procedures.

Wavelength and orientation dependent capture of light by diatom frustule nanostructures

The ecological success of diatoms is emphasized by regular blooms of many different species in all aquatic systems, but the reason behind their success is not fully understood. A special feature of the diatom cell is the frustule, a nano-patterned cell encasement made of amorphous biosilica. The optical properties of a cleaned single valve (one half of a frustule) from the diatom Coscinodiscus centralis were studied using confocal micro-spectroscopy. A photonic crystal function in the frustule was observed, and analysis of the hyperspectral mapping revealed an enhancement of transmitted light around 636 and 663 nm. These wavelengths match the absorption maxima of chlorophyll a and c, respectively. Additionally, we demonstrate that a highly efficient light trapping mechanism occurred, resulting from strong asymmetry between the cribrum and foramen pseudo-periodic structures. This effect may prevent transmitted light from being backscattered and in turn enhance the light absorption. Based on our results, we hypothesize that the multi-scaled layered structure of the frustule improves photosynthetic efficiency by these three mechanisms. The optical properties of the frustule described here may contribute to the ecological success of diatoms in both lentic and marine ecosystems, and should be studies further in vivo.

High-resolution magic angle spinning NMR analysis of whole cells of Chaetoceros muelleri (Bacillariophyceae) and comparison with 13C-NMR and distortionless enhancement by polarization transfer 13C-NMR analysis of lipophilic extracts

Lipid composition in extracted samples of Chaetoceros muelleri Lemmermann was studied with 13C‐NMR and distortionless enhancement by polarization transfer (DEPT) 13C‐NMR, resulting in well‐resolved 13C‐NMR spectra with characteristic resonance signals from carboxylic, olefinic, glyceryl, methylene, and methyl groups. The application of a DEPT pulse sequence aided in the assignment of methylene and methine groups. Resonance signals were compared with literature references, and signal assignment included important unsaturated fatty acids such as eicosapentaenoic and docosahexaenoic and also phospholipids and glycerols. Results from the extracted samples were used to assign resonance signals in a high‐resolution magic angle spinning (HR MAS) DEPT 13C spectrum from whole cells of C. muelleri. The NMR analysis on whole cells yielded equally good information on fatty acids and also revealed signals from carbohydrates and amino acids. Broad resonance signals and peak overlapping can be a problem in whole cell analysis, but we found that application of HR MAS gave a well‐resolved spectrum. The chemical shift of metabolites in an NMR spectrum depends on the actual environment of nuclei during analysis, and some differences could therefore be expected between extracted and whole cell samples. The shift differences were small, and assignment from analysis of lipophilic extract could be used to identify peaks in the whole cell spectrum. HR MAS 13C‐NMR therefore offers a possibility for broad‐range metabolic profiling directly on whole cells, simultaneously detecting metabolites that are otherwise not detected in the same analytical set up and avoiding tedious extraction procedures.

1H NMR metabolic profiling of cod (Gadus morhua) larvae: potential effects of temperature and diet composition during early developmental stages

ABSTRACT Marine aquaculture offers a great source of protein for the increasing human population, and farming of, for example, Atlantic salmon is a global industry. Atlantic cod farming however, is an example of a promising industry where the potential is not yet realized. Research has revealed that a major bottleneck to successful farming of cod is poor quality of the larvae and juveniles. A large research program was designed to increase our understanding of how environmental factors such as temperature and nutrition affects cod larvae development. Data on larvae growth and development were used together with nuclear magnetic resonance. The NMR data indicated that the temperature influenced the metabolome of the larvae; differences were related to osmolytes such as betaine/TMAO, the amino acid taurine, and creatine and lactate which reflect muscle activity. The larvae were fed Artemia from stage 2, and this was probably reflected in a high taurine content of older larvae. Larvae fed with copepods in the nutrition experiment also displayed a high taurine content, together with higher creatine and betaine/TMAO content. Data on the cod larvae metabolome should be coupled to data on gene expression, in order to identify events which are regulated on the genetic level versus regulation resulting from temperature or nutrition during development, to fully understand how the environment affects larval development. Summary: Metabolomic ‘snapshots’ from developing cod larvae reflect the temperature and diet experienced from hatching to juvenile and provide insight into how important processes such as osmoregulation and muscle development might be affected.

Dynamic responses to silicon in Thalasiossira pseudonana - Identification, characterisation and classification of signature genes and their corresponding protein motifs

The diatom cell wall, or frustule, is a highly complex, three-dimensional structure consisting of nanopatterned silica as well as proteins and other organic components. While some key components have been identified, knowledge on frustule biosynthesis is still fragmented. The model diatom Thalassiosira pseudonana was subjected to silicon (Si) shift-up and shift-down situations. Cellular and molecular signatures, dynamic changes and co-regulated clusters representing the hallmarks of cellular and molecular responses to changing Si availabilities were characterised. Ten new proteins with silaffin-like motifs, two kinases and a novel family of putatively frustule-associated transmembrane proteins induced by Si shift-up with a possible role in frustule biosynthesis were identified. A separate cluster analysis performed on all significantly regulated silaffin-like proteins (SFLPs), as well as silaffin-like motifs, resulted in the classification of silaffins, cingulins and SFLPs into distinct clusters. A majority of the genes in the Si-responsive clusters are highly divergent, but positive selection does not seem to be the driver behind this variability. This study provides a high-resolution map over transcriptional responses to changes in Si availability in T. pseudonana. Hallmark Si-responsive genes are identified, characteristic motifs and domains are classified, and taxonomic and evolutionary implications outlined and discussed.