The lipid composition of Euglena gracilis middle plastid membrane resembles that of primary plastid envelopes.

Euglena gracilis is a photosynthetic flagellate possessing chlorophyte-derived secondary plastids that are enclosed by only three enveloping membranes, unlike most secondary plastids, which are surrounded by four membranes. It has generally been assumed that the two innermost E. gracilis plastid envelopes originated from the primary plastid, while the outermost is of eukaryotic origin. It was suggested that nucleus-encoded plastid proteins pass through the middle and innermost plastid envelopes of E. gracilis by machinery homologous to the translocons of outer (TOC) and inner (TIC) chloroplast membranes, respectively. Although recent genomic, transcriptomic, and proteomic data proved the presence of a reduced form of the TIC complex, they failed to identify any TOC homologues, which raised the question of the origin of E. gracilis's middle plastid envelope. Here, we compared the lipid composition of whole cells of the pigmented E. gracilis strain Z and two bleached mutants that lack detectable plastid structures, W10BSmL and WgmZOflL. We determined the lipid composition of E. gracilis strain Z mitochondria and plastids, and of plastid subfractions (thylakoids and envelopes), using HPLC HR-MS/MS, TLC, and GC-FID analytical techniques. Phosphoglycerolipids are the main structural lipids in mitochondria, while glycosyldiacylglycerols (GDAGs) are the major structural lipids of plastids and also predominate in extracts of whole mixotrophic cells. GDAGs were detected in both bleached mutants, indicating that mutant cells retain some plastid remnants. Additionally, we discuss the origin of the E. gracilis middle plastid envelope based on its lipid composition.