Integrated systems biology and imaging of the smallest free-living eukaryote Ostreococcus tauri

Abstract

1Ostreococcus tauri is an ancient phototrophic microalgae that possesses favorable 2 genetic and cellular characteristics for reductionist studies probing biosystem design and 3 dynamics. Here multimodal bioimaging and multi-omics techniques were combined to 4 interrogate O. tauri cellular changes in response to variations in bioavailable nitrogen and 5 carbon ratios. Confocal microscopy, stimulated Raman scattering, and cryo-soft x-ray 6 tomography revealed whole cell ultrastructural dynamics and composition while proteomic and 7 lipidomic profiling captured changes at the molecular and macromolecular scale. 8Despite several energy dense long-chain triacylglycerol lipids showing more than 40-fold 9 higher abundance under N deprivation, only a few proteins directly associated with lipid 10 biogenesis showed significant expression changes. However, the entire pathway for starch 11 granule biosynthesis was highly upregulated suggesting much of the cellular energy is 12 preferentially directed towards starch over lipid accumulation. Additionally, three of the five most 13 downregulated and five of the ten most upregulated proteins during severe nitrogen depletion 14 were unnamed protein products that warrant additional biochemical analysis and functional 15 annotation to control carbon transformation dynamics in this smallest eukaryote. 16 17 19 triacylglycerol, lipid, starch, carbon transformation, lipid droplet, nitrogen scavenging, soft x-ray 20 tomography, stimulated raman scattering microscopy, fluorescence microscopy 21 22 phylogenetically diverse and exhibit varying cellular phenotypes that naturally produce high 26 value metabolites, proteins, carbohydrates and energy dense lipids that can be exploited for a 27 wide array of industrial applications 1-3 . Due to their high photosynthetic efficiency for energy 28 conversion, and minimal growth requirements consisting of sustainable resources such as 29 marine or brackish media, light, CO2 and trace vitamins, microalgae are prime bioproduction 30 platforms 4,5 . 31 Triacylglycerol (TAG) lipids are significantly enhanced when microalgae are subjected to 32 cellular stressors such as light, temperature, and nutrient deprivation 6 . TAG lipids possess 33 nonpolar character and are stored in anhydrous, high-density organelles called lipid bodies, 34 which are desirable for industrial lipid feedstock applications 7 . For other oleaginous algae, TAG 35production can be triggered by nutrient deprivation of iron, sulfur, nitrogen, phosphate, or silicon 36 6 . In most eukaryotes, combinatorial reduction of these nutrients results in altered levels of 37 growth-associated structural lipids (phospholipids) and energy storage lipids (TAG) products 8,9 . 38 Unfortunately, in most cases starvation or deprivation can be detrimental to cell viability and 39 overall growth capacity thereby limiting cell biomass yields needed for viable lipid feedstock 40 industrial applications 10 . While several reports have shown that supplementing additional C 41 sources when combined wit...

Circadian protein regulation in the green lineage I. A phospho-dawn of protein modification anticipates light onset in the picoeukaryoteO. tauri

“…A preprint coincident with our first report showed that three of these night-expressed, prasinophyte-specific proteins accumulated strongly in O. tauri when the growth medium was depleted of nitrogen, under LD cycles (Smallwood et al, 2018a). The most-depleted protein in their conditions was the same starch synthase (ostta06g02940) that fell most in abundance under our prolonged dark treatment.…”

A phospho-dawn of protein modification anticipates light onset in the picoeukaryoteOstreococcus tauri

“…The third most depleted protein in their conditions was the same starch synthase (ostta06g02940) that fell most in abundance under our prolonged dark treatment. Smallwood et al also showed that O. tauri forms both intracellular and extracellular lipid droplets under their conditions ( Smallwood et al , 2018a , Preprint, 2018b , Preprint). It is possible that sorbitol and glycerol from our medium were metabolized to lipids, and that the night-expressed proteins contributed to that process (see the Discussion).…”

“…A pre-print ( Smallwood et al ., 2018a , Preprint) coincident with our first report ( Noordally et al , 2018 , Preprint) showed that three of the night-expressed, prasinophyte-specific proteins accumulated strongly in O. tauri under LD cycles when the growth medium was depleted of nitrogen, particularly if carbon availability was also increased (ostta03g04500 accumulated most; ostta09g00670, third; ostta02g03680, fifth). The third most depleted protein in their conditions was the same starch synthase (ostta06g02940) that fell most in abundance under our prolonged dark treatment.…”

Structure Of Eukaryotic Type III Glutamine Synthetase Lacking Ring-Ring Quaternary Contacts

“…To determine which annotation of GSIII is natively expressed in O. tauri , we reviewed data from our prior global and phosphoproteomic analyses 38 of wild-type cultures grown in Keller marine media under both normal and nitrogen depleted growth conditions. Under nitrogen deprivation, the GSIII protein was the 10 th highest upregulated protein detected in the published datasets, suggesting its potentially critical role in nitrogen metabolism and recycling.…”

“…Class II GSs (GSII) are considered eukaryotic-specific but instances of them are also found in some families of bacteria 13,14 . Individual GSII subunits are slightly smaller in size (around [35][36][37][38][39][40], where ten subunits participate in the protein complex formation, forming two stacked pentameric rings with D5 symmetry 12,15,16 (Fig. S1).…”