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Abstract Lipid structures affect membrane biophysical properties such as thickness, stability, permeability, curvature, fluidity, asymmetry, and interdigitation, contributing to membrane function. Sphingolipids are abundant in plant endomembranes and plasma membranes (PMs) and comprise four classes: ceramides, hydroxyceramides, glucosylceramides, and glycosylinositolphosphoceramides (GIPCs). They constitute an array of chemical structures whose distribution in plant membranes is unknown. With the aim of describing the hydrophobic portion of sphingolipids, 18 preparations from microsomal (MIC), vacuolar (VM), PM, and detergent-resistant membranes (DRM) were isolated from Arabidopsis (Arabidopsis thaliana) leaves. Sphingolipid species, encompassing pairing of long-chain bases and fatty acids, were identified and quantified in these membranes. Sphingolipid concentrations were compared using univariate and multivariate analysis to assess sphingolipid diversity, abundance, and predominance across membranes. The four sphingolipid classes were present at different levels in each membrane: VM was enriched in glucosylceramides, hydroxyceramides, and GIPCs; PM in GIPCs, in agreement with their key role in signal recognition and sensing; and DRM in GIPCs, as reported by their function in nanodomain formation. While a total of 84 sphingolipid species was identified in MIC, VM, PM, and DRM, only 34 were selectively distributed in the four membrane types. Conversely, every membrane contained a different number of predominant species (11 in VM, 6 in PM, and 17 in DRM). This study reveals that MIC, VM, PM, and DRM contain the same set of sphingolipid species but every membrane source contains its own specific assortment based on the proportion of sphingolipid classes and on the predominance of individual species. 

Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. InPhyscomitrella patens, 4‐hydroxysphinganine (phytosphingosine, t18:0) is the predominant sphingolipid long‐chain base (LCB). To assess the functional significance of t18:0, CRISPR‐Cas9 mutagenesis was used to generate mutant lines lacking the soleSPHINGOID BASE HYDROXYLASE(SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content insbhprotonemata was 2.4‐fold higher than in wild‐type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t18:0 indicated modest alterations in acyl‐chain pairing with d18:0 in GIPCs and ceramides, but dramatic alterations in acyl‐chain pairing in glucosylceramides, in which 4,8‐sphingadienine (d18:2) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. Thesbhlines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t17:0, the endogenous sphingolipid content ofsbhlines decreased to wild‐type levels, and the mutants exhibited phenotypes more similar to wild‐type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyteP. patensand angiospermA. thaliana.