Genome-Scale Model Reveals Metabolic Basis of Biomass Partitioning in a Model Diatom.

Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curated reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. The model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications.

Oxidative pathways in response to polyunsaturated aldehydes in the marine diatom Skeletonema marinoi (Bacillariophyceae).

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR-derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA-exposed S. marinoi cells were treated with the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an opposite response was observed, with an increase in DHR-derived green fluorescence. A higher DHR-derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2-methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O2 (•) generation and SOD activity in H2 O2 formation (with downstream ROS generation dependent from H2 O2 ) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA-exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O2 (•-) and H2 O2 . This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.

The effect of polyunsaturated aldehydes on Skeletonema marinoi (Bacillariophyceae): the involvement of reactive oxygen species and nitric oxide.

Nitric oxide (NO) and reactive oxygen species (ROS) production was investigated in the marine diatom, Skeletonema marinoi (SM), exposed to 2E,4E/Z-decadienal (DECA), 2E,4E/Z-octadienal (OCTA), 2E,4E/Z-heptadienal (HEPTA) and a mix of these last two (MIX). When exposed to polyunsaturated aldehydes (PUA), a decrease of NO was observed, proportional to the PUA concentration (85% of the initial level after 180 min with 66 µM DECA). Only OCTA, HEPTA and MIX induced a parallel increase of ROS, the highest (2.9-times the control) with OCTA concentrations twice the EC50 for growth at 24 h (20 µM). The synthesis of carotenoids belonging to the xanthophyll cycle (XC) was enhanced during exposure, suggesting their antioxidant activity. Our data provide evidence that specific pathways exist as a reaction to PUA and that they depend upon the PUA used and/or the diatom species. In fact, Phaeodactylum tricornutum (PT) produces NO in response to DECA, but not to OCTA. We advance the hypothesis that SM perceives OCTA and HEPTA as intra-population infochemicals (as it produces PUA), while PT (non-PUA producing species) perceives them as allelochemicals. The ability to produce and to use PUA as infochemicals may underlie ecological traits of different diatom species and modulate ecological success in natural communities.