Molecular characterization and targeted quantitative profiling of the sphingolipidome in rice

Abstract

Recent advances in comprehensive metabolite profiling techniques, the foundation of metabolomics, is facilitating our understanding of the functions, regulation and complex networks of various metabolites in organisms. Here, we report a quantitative metabolomics technique for complex plant sphingolipids, composed of various polar head groups as well as structural isomers of hydrophobic ceramide moieties. Rice (Oryza sativa L.) was used as an experimental model of monocotyledonous plants and has been demonstrated to possess a highly complex sphingolipidome including hundreds of molecular species with a wide range of abundance. We established a high-throughput scheme for lipid preparation and mass spectrometry-based characterization of complex sphingolipid structures, which provided basic information to create a comprehensive theoretical library for targeted quantitative profiling of complex sphingolipids in rice. The established sphingolipidomic approach combined with multivariate analyses of the large dataset obtained clearly showed that different classes of rice sphingolipids, particularly including subclasses of glycosylinositol phosphoceramide with various sugar-chain head groups, are distributed with distinct quantitative profiles in various rice tissues, indicating tissue-dependent metabolism and biological functions of the lipid classes and subclasses. The sphingolipidomic analysis also highlighted that disruption of a lipid-associated gene causes a typical sphingolipidomic change in a gene-dependent manner. These results clearly support the utility of the sphingolipidomic approach in application to wide screening of sphingolipid-metabolic phenotypes as well as deeper investigation of metabolism and biological functions of complex sphingolipid species in plants.

Plant-Unique cis/trans Isomerism of Long-Chain Base Unsaturation is Selectively Required for Aluminum Tolerance Resulting from Glucosylceramide-Dependent Plasma Membrane Fluidity

“…Figure 4 shows profiles of the two major glycosphingolipid classes, GlcCer (A) and GIPC (B). Consistent with previous reports, both classes displayed distinct LCB compositions in their ceramide moieties: GlcCer primarily contained ∆8-unsaturated LCBs but GIPC was mainly composed of saturated t18:0 ( Figure 4; [25]). In OsSLD-OX and AtSLD-OX, t18:0 decreased and t18:1 increased in GIPC, whereas t18:1-containing GlcCer was only slightly elevated.…”

“…In addition, the cis/trans ratios of SLD products are highly diverse and apparently determine the isomeric compositions of the final product glycosphingolipids in each plant [5,23]. For example, SLDs from Arabidopsis and many dicots predominantly generate the trans bond rather than the cis, while enzymes from monocots preferentially generate the cis configuration, roughly consistent with the isomer ratios in those plants [5,[23][24][25].…”

“…In addition, LCB structures are thought to play an important role in the selective distribution of ceramide backbones into the two major classes, GlcCer and GIPC [5]. GIPC contains negative charges in the head group [3,25] that are possible targets of Al 3+ binding on the surface of the plasma membrane as reported in phospholipids [27], causing aberrant membrane fluidity and function. To our knowledge, however, there is no report addressing the class-specific association of sphingolipids in aluminum tolerance.…”

“…In addition, the two glycosphingolipid classes have distinct ceramide moieties in the LCB ∆8 structure. GlcCer selectively incorporates ∆8 unsaturated LCBs at a higher cis/trans ratio, while GIPC prefers ∆8 trans-unsaturated or saturated species ( [3,25,35]; Figure S1). How are these unique classes associated with aluminum toxicity and tolerance thorough LCB unsaturation and membrane fluidity?…”

Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses and Reduces Cellulose Content in Arabidopsis

“…For the sphingolipidomics, total sphingolipids were extracted from dried (30 to 50 mg) or fresh (100 to 200 mg) plant materials and subjected to lipid profiling by LC-MS/MS as described previously (Nagano et al, 2014). GIPCs were quantified according to Ishikawa et al (2016).…”