Stepwise Biogenesis of Subpopulations of Lipid Droplets in Nitrogen Starved Phaeodactylum tricornutum Cells

Abstract

Diatoms are unicellular heterokonts, living in oceans and freshwaters, exposed to frequent environmental variations. They have a sophisticated membrane compartmentalization and are bounded by a siliceous cell-wall. Formation of lipid droplets (LDs), filled with triacylglycerol (TAG), is a common response to stress. The proteome of mature-LDs from Phaeodactylum tricornutum highlighted the lack of proteins involved in early-LD formation, TAG biosynthesis or LD-toLD connections. These features suggest that cytosolic LDs might reach a size limit. We analyzed the dynamics of LD formation in P. tricornutum (Pt1 8.6; CCAP 1055/1) during 7 days of nitrogen starvation, by monitoring TAG by mass spectrometry-based lipidomics, and LD radius using epifluorescence microscopy and pulse field gradient nuclear magnetic resonance. We confirmed that mature LDs reach a maximal size. Based on pulse field gradient nuclear magnetic resonance, we did not detect any LD-LD fusion. Three LD subpopulations were produced, each with a different maximal size, larger-sized LDs (radius 0.675 ± 0.125 µm) being generated first. Mathematical modeling showed how smaller LDs are produced once larger LDs have reached their maximum radius. In a mutant line having larger cells, the maximal size of the first LD subpopulation was higher (0.941 ± 0.169 µm), while the principle of stepwise formation of distinct LD populations was maintained. Results suggest that LD size is determined by available cytosolic space and sensing of an optimal size reached in the previous LD subpopulation. Future perspectives include the unraveling of LD-size control mechanisms upon nitrogen shortage. This study also provides novel prospects for the optimization of oleaginous microalgae for biotechnological applications.

High Resolution Proteome of Lipid Droplets Isolated from the Pennate Diatom Phaeodactylum tricornutum (Bacillariophyceae) Strain pt4 provides mechanistic insights into complex intracellular coordination during nitrogen deprivation

“…This observation has been corroborated by a recently published morphometric analysis of Pt1 LDs (Jaussaud et al. 2020), as well as our morphometric analysis of Pt4, discussed later in this paper. Furthermore, our preliminary observations suggested that Pt4 appeared to have higher yields of LDs from cells disrupted by hypotonic shock, perhaps because they are more amenable to this particular cell disruption method, and potentially related to differences in membrane lipid composition (Steinrücken et al.…”

“…This is particularly curious, since TAG biosynthesis enzymes have been shown to translocate from the ER to LDs in other organisms (Wilfling et al 2013a). In P. tricornutum strain Pt1, LDs were reported to reach a maximum size after three days of N starvation (Jaussaud et al 2020), and by the seventh day of N starvation, no TAG biosynthesis enzymes were identified in the LD proteome (Lupette et al 2019). In contrast, we found that LDs PHAEODACTYLUM LIPID DROPLET PROTEOME continued to expand throughout seven days of N starvation.…”

“…In P. tricornutum strain Pt1, LDs were reported to reach a maximum size after three days of N starvation (Jaussaud et al. 2020), and by the seventh day of N starvation, no TAG biosynthesis enzymes were identified in the LD proteome (Lupette et al. 2019).…”

Lipid Droplets in Unicellular Photosynthetic Stramenopiles

“…The architecture of the secondary plastids is unique, and the outermost membrane, the EpM, is in continuity with the ER (see above). Electron microscopy analyses have shown very close contacts between the plastid and the LD in P. tricornutum ( Flori et al, 2016 ; Lupette et al, 2019 ; Jaussaud et al, 2020 ). Moreover, the lipid monolayer surrounding the LD includes the plastid glycolipid SQDG as well as PC and the betaine lipid DGTA ( Lupette et al, 2019 ) (see above) that is considered to be synthesized in the ER.…”

Comprehensive Utilization of Marine Microalgae for Enhanced Co-Production of Multiple Compounds

“…Thus, a two-stage cultivation strategy, with sufficient nutrient for cell growth in the first stage, and nutrient limitation for metabolites accumulation during the second stage, has been used to promote the co-production of multiple compounds [114]. For example, a nitrogen sufficient/deficient strategy was used to increase TAG accumulation in Isochrysis zhangjiangensis [109] and P. tricornutum [110]. In addition, nutrient sufficient/deficient strategy has been widely used in secondary carotenoid production, such as β-carotene [108].…”