Plasma and vacuolar membrane sphingolipidomes: composition and insights on the role of main molecular species

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.

Figure 1

Experimental approach followed for the analysis of membrane sphingolipidomes. Arabidopsis (A. thaliana; Col-0) leaves from 11-week-old plants were used to obtain the crude membrane fraction, microsomes (MIC). From this, VM and PM were purified. PM fractions were obtained by two methods, phase partitioning, and free-flow electrophoresis. DRM fractions were obtained from the PMs isolated by two-phase partitioning. Every membrane preparation was independently treated and extracted for sphingolipid analysis by HPLC/ESI-MS/MS. Thus, the 18 independent membrane preparations are named 18 biological replicates and are composed of six MIC preparations, three VM preparations, three PM preparations obtained by two-phase partitioning, three PM preparations obtained by FFZE, and three DRM preparations. Every biological replicate was used for one sphingolipid extraction and sphingolipid analysis (one technical replicate). However, only for one biological sample from each type of membrane, two extractions were performed (two technical replicates). Therefore, a total of 23 technical replicates were obtained as shown. The different statistical tests applied to the data are indicated.

Figure 2

Purity assessment of MIC, VM, PM, and DRM fractions from Arabidopsis leaves. A, Transmission electron micrographs from MIC, VM, PM, and DRM preparations from Arabidopsis leaves. MIC, VM, PM, and DRM pellets were fixed and processed as indicated in the Materials and Methods. B, Immunodetection of the PM H⁺-ATPase and a PM aquaporin, the Na⁺/H⁺ antiporter from the VM, the AOX from the mitochondrial inner membrane, and the sterol methyltransferase 1 from ER. Membrane proteins were separated by SDS-PAGE and detected by immunoblot. C, Protein loading was determined by Coomassie blue staining of a replicate gel, wherein equal quantities of protein were loaded in the lanes. MIC, microsomal fraction.

Figure 3

Sphingolipid content and sphingolipid class distribution from MIC, VM, PM, and DRM of Arabidopsis leaves. A, Representation of the structure of the main sphingolipid classes studied in this work. Cers, ceramides; hCers, hydroxyceramides; GlcCers, glucosylceramides; GIPCs, glycosylinositolphosphoceramides; FA, fatty acid; hFA, hydroxylated fatty acid; LCB, long-chain base. B, Total sphingolipid content from MIC, VM, PM, and DRM preparations. C, Content of every sphingolipid class in MIC, VM, PM, and DRM. Membrane types are indicated in colors (light gray for MIC, magenta for VM, dark blue for PM, and dark grey for DRM). D, Distribution of sphingolipid classes within every membrane type. Values are expressed as mean ± SE from 4 to 8 technical replicates depending on the membrane source from the independent biological preparations (see Figure 1). Different lowercase labels in bars indicate statistical differences (i.e., in panel B, VM and PM labeled as “b” were not statistically different among them, while both VM and PM are statistically different to MIC labeled as “a” and to DRM labeled as “c”). One-way ANOVA with Fisher’s post hoc test for multiple comparisons, P < 0.05 was performed.

Figure 4

Multivariate analysis and heatmap plot of the sphingolipidome profiles from MIC, VM, PM, and DRM of Arabidopsis leaves. A, Partial least squares-discriminant analysis (PLS-DA) of sphingolipid profiles from the independent preparations of MIC, VM, PM, and DRM obtained from Arabidopsis leaves. B, Comparison of global sphingolipidomes from MIC, VM, PM, and DRM. Heat map visualization shows the standardized (z-scores) concentrations of individual sphingolipid species from mol %. Hierarchical clustering of the sphingolipid classes was used to group similar profiles using Euclidean distances and Ward clustering.The scale from 1 to −1 represents the number of standard deviations from the mean. Blue color indicates lower concentrations than average and red color indicates higher concentrations than average. In B, average values from the technical replicates were considered for each membrane preparation (7, 4, 8, and 4 for MIC, VM, PM, and DRM, respectively). These analyses required the processing of a total of 1,932 values from LCBs and FAs (mol %) that yielded 84 paired sphingolipid species (a LCB bound to a FA each) from the 23 technical replicates. The sphingolipid classes were Cers, ceramides; hCers, hydroxyceramides; GlcCers, glucosylceramides; GIPCs, glycosylinositolphosphoceramides.

Figure 5

Distribution of molecular sphingolipid species according to their diversity and abundance in MIC, VM, PM, and DRM from Arabidopsis leaves. A. Distribution of Cer species in MIC, VM, PM, and DRM. Left pie chart includes the highly abundant species and right pie chart includes the low abundant species. Bar graphs at the right side indicate the number of low and high abundant species. Structures of highly abundant species in three different abundance intervals are depicted in colors according to the membrane type (light gray for MIC, magenta for VM, dark blue for PM, and dark grey for DRM). B–D, Illustrate the same but for hCers, GlcCers, and GIPCs, respectively. The abundance of sphingolipid species is expressed in mol % from 4 to 8 technical replicates depending on the membrane source (see Figure 1).

Figure 6

Differential distribution of individual species of Cers and hCers among the MIC, VM, PM, and DRM from Arabidopsis leaves. A and B, Cer individual species. C–E, hCer individual species. Values are expressed as mean ± SE from 4 to 8 replicates depending on the membrane source (see Figure 1). Different lowercase labels between bars indicate statistical differences as described in Figure 3.

Figure 7

Differential distribution of GlcCer and GIPC individual species among the MIC, VM, PM, and DRM from Arabidopsis leaves. A, GlcCer individual species. B and C, GIPC individual species. Values are expressed as mean ± SE from 4 to 8 replicates depending on the membrane source (see Figure 1). Different lowercase labels between bars indicate statistical differences as described in Figure 3.