Advanced LC-MS/MS Techniques Dissecting Diverse Isomers of Plant Sphingolipid Species

Sphingolipids is characterized to be composed of a unique fatty amino alcohol, so-called long-chain base (LCB) or sphingoid base, most of which are linked to a fatty acid via amide bond to form ceramide and more complex sphingolipids following further glycosylation and/or phosphorylation of the ceramide backbone (Figure 1). Sphingolipids are conserved ubiquitously in eukaryotes and a few species in prokaryotes. In mammals, most sphingolipids contain sphingosines with transunsaturation at the Δ4 position (Figure 1). Alternatively, 4-hydroxylated LCBs, so-called phytosphingosines, are observed in limited tissues in mammals [1-3] but known as major components in plants and fungi: in these organisms, notably, 4-unsaturated or 4-hydroxylated LCBs are often further unsaturated at the Δ8 position with cis/trans isomerism [4]. The Δ8 unsaturation is generated by sphingolipidΔ8desaturase (SLD) that is conserved widely in plants and fungi, although model yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe exceptionally lack this enzyme in their genome. In addition, fungal SLDs seem to introduce a transspecific double bond, whereas plant counterparts produce both cis and trans isomers at various ratios according to plant species as well as to sphingolipid classes even in a certain species [5], implying that the stereoisomerism of LCB Δ8 unsaturation has been developed, especially in plants, along with specific biochemical/physiological functions. Recent studies suggested that LCB Δ8 unsaturation is not always essential for normal growth but plays crucial roles in some situations. Ryan et al. [6] isolated a SLD homolog from acidic soil-compatible plant Stylosanthes hamata as an Al3+tolerance-conferring gene when ectopically expressed in S. cerevisiae. The authors also demonstrated that overexpression of S. hamata SLD in Arabidopsis plants increases cis/trans ratio of tri-hydroxy species, i.e., t18:1(c8)/t18:1(t8), and enhances Al3+ tolerance. Although the authors did not address what kinds of sphingolipid classes as shown in Figure 1 are involved in the Δ8 unsaturation-mediated Al3+tolerance in their published work, accumulated evidences implicate that glucosylceramides (GlcCers) is a possible candidate: biochemical studies have revealed that plant GlcCers generally prefer higher cis/transratio compared to other lipid classes, e.g. free ceramides and glycosyl inositol phosphoceramdes (GIPCs) [710]. In fact, Arabidopsis sld mutant lacking Δ8 unsaturation of LCB moieties showed drastic decreases in GlcCer species but neither in Cer nor GIPC [11]. S. cerevisiae and S. pombelack not only LCB desaturases but also GlcCer synthase, which might indicate evolutionary relationship between LCB unsaturation and GlcCer synthesis. In addition, loss of endogenous GlcCer synthase leads to higher sensitivity to Al3+ toxicity in Kluyveromyceslactis (Imai et al. unpublished data). These insights suggest that sphingolipid Δ8 unsaturation is closely associated with GlcCer synthesis, including cis isomers preferably in some cases, which has a crucial role in Al3+ tolerance based on conserved mechanisms in plants and fungi. Another physiological function of Δ8 unsaturation is relation to cold tolerance, which was first proposed from comparative analysis of LCB composition in GlcCer in several grapevine species showing different cold sensitivity [12]. A recent reverse genetic study on Arabidopsis sld1mutants supported implication of Δ8 unsaturation of GlcCer LCB moieties in cold tolerance [11]. Δ8 unsaturation-deficient sld1mutants showed not only lower GlcCer contents but also elevated sensitivity to cold stress. The authors clearly demonstrated the altered sphingolipid profiles by LC-MS/MS-based sphingolipidomic analysis, which has become an essential tool for sphingolipid studies in plants established by several groups [9,13,14]. For example, in the established LC-MS/MS analyses using a reverse-phase C18 column, sphingolipid species are separated according to their structural identities, i.e., 1) structures of polar head group; 2) carbon length, n-9 double bond and 2-hydroxylation of fatty acyl moieties; 3) C4-hydroxylation and Δ4/Δ8 double bonds