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...