The architecture of lipid droplets in the diatom Phaeodactylum tricornutum

Abstract

Diatoms are a major phylum of phytoplankton biodiversity and a resource considered for biotechnological developments, as feedstock for biofuels and applications ranging from food, human health or green chemistry. They contain a secondary plastid limited by four membranes, the outermost one being connected with the endoplasmic reticulum (ER). Upon nitrogen stress, diatoms reallocate carbon to triacylglycerol storage inside lipid droplets (LDs). The comprehensive glycerolipid and sterol composition and the architecture of diatom LDs are unknown. In Phaeodactylum tricornutum, LDs are in contact with plastid, mitochondria and uncharacterized endomembranes. We purified LDs from nitrogen-starved P. tricornutum cells to high purity level (99 mol% triacylglycerol of total glycerolipids). We used the Stramenopile Lipid Droplet Protein (StLDP) as a previoulsy validated marker for the identity of P. tricornutum LD. Amphipathic lipids surrounding LDs consist of a betaine lipid, diacylglycerylhydroxymethyltrimethyl-β-alanine (0.4 mol%); sulfoquinovosyldiacylglycerol (0.35 mol%); phosphatidylcholine (0.15 mol%) and one sterol, brassicasterol. By contrast with whole cell extracts, the betaine lipid from LDs only contains eicosapentaenoic acid paired with palmitoleic or palmitolenic acids. This polar lipid composition suggests a budding of LDs from the cytosolic leaflet of the plastid outermost membrane. LD pigments reveal a specific accumulation of β-carotene. The LD proteome obtained from three independent biological replicates, based on stringent filtering of extracted data, and following subtraction of proteins downregulated by nitrogen starvation, highlights a core proteome of 86 proteins, including StLDP. LD-associated proteins suggest connections with vesicular trafficking (coatomer, clathrin), cytoskeleton, plastid and mitochondria. Unsuspected LD-associated function include protein synthesis (ribosomes), folding (chaperones), posttranslational modifications and quality control (ubiquitination and ERAD pathway), possibly preparing translation of specific mRNAs. The detection of histone proteins, as previously demonstrated in drosophila embryo LDs, also suggests the storage of nucleosome components, preparing cell division and chromatin packaging, when cells are not stressed anymore.

A Review of Diatom Lipid Droplets

“…Various species of green algae sequester carotenoids in LDs, which may shield the photosynthetic apparatus from excessive light, reduce reactive oxygen species, serve as a sink for excess photosynthates or protect LD lipids from oxidation. Evidence of several pigments in P. tricornutum and F. solaris LDs has been found, including beta-carotene and fucoxanthin, as well as fucoxanthin-chlorophyll a/c binding proteins [37][38][39]. Our lab has made similar observations (unpublished) but the presence and function of carotenoids or other isoprenoids, in diatoms has not been explored in detail yet.…”

“…TAGs store large amounts of energy, carbon and membrane components, while simultaneously shielding the cell from the potential cytotoxicity of free fatty acids [34][35][36]. Thin-layer chromatography of LDs isolated from P. tricornutum confirms they are indeed composed mostly of TAG [37]; however recent lipidomic analysis has suggested that some sterols are present in lower abundance [38].…”

“…In most eukaryotes, the phospholipid monolayer of LDs is typically composed of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) [49], however in microalgae other lipid classes have been observed, including chloroplast glycerolipids and betaine lipids [50,51]. Lipidomic analysis of nitrogen starved P. tricornutum (Pt.1) identified the major polar lipids to be sulfoquinovosyldiacylglycerol (SQDG), PC and the betaine lipid diacylglycerylhydroxymethyltrimethyl-β-alanine (DGTA) [38]. The polar lipid composition of the LD membrane may affect which proteins bind to it [52].…”

“…Administration of the inhibitor chloroquine, which impairs the fusion of autophagosomes with lysosomes [146], suppressed degradation of mature LDs [39]. Clathrin was identified in the LD proteomes of both F. solaris and P. tricornutum [38,39,147]. During recovery from nutrient starvation, LDs shrink in size due to lipolysis or lipophagy.…”

“…So far, diatom LD proteins have only been isolated from weakly silicified marine raphid pennate species from the order Naviculales, F. solaris and P. tricornutum [37][38][39]147,190,191]. Although centrics and pennates are genetically quite disparate, similarities in the metabolic responses of T. pseudonana [89] and P. tricornutum [85] to N starvation suggests that we could expect some general similarities between the LD proteomes of the Naviculales and other diatoms.…”