Identification of a sphingolipid‐specific phospholipase D activity associated with the generation of phytoceramide‐1‐phosphate in cabbage leaves

Abstract

The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage (Brassica oleracea) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI-TOF MS as an adduct with Phos-tag, a phosphate-capture zinc complex. Various α-hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC-MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4-hydroxysphingenine (dehydrophytosphingosine)-1-phosphate by MALDI-TOF MS with Phos-tag. From these results, lipid X was determined to be phytoceramide-1-phosphate (PC1P) with an α-hydroxy fatty acid. When cabbage homogenates were incubated, PC1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (GIPC). The formation of PC1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin. Arabidopsis thaliana also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC1P in brassicaceous plants.

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

“…Although it has been reported that Brassicacea extracts (from cabbage and Arabidopsis) have GIPC-specific phosphatase activity (Tanaka et al, 2013), as yet the gene encoding the enzyme responsible has not been cloned. Identification of this enzyme will be very beneficial for GIPC research, enabling NMR characterization of the released glycan.…”

Molecular characterization and targeted quantitative profiling of the sphingolipidome in rice

“…Nipponbare, was cultivated on soil at 28°C under 12 h dark/12 h light cycles. To inactivate potent sphingolipid‐degrading enzymes present in plant tissues (Tanaka et al ., ), harvested tissues (100–300 mg) were immediately immersed in isopropanol prewarmed at 80°C and kept for 10 min to inactivate lipid‐degrading enzymes, followed by storage at −30°C until use. Seeds obtained from heterologous lines of oni mutants were germinated and grown in tapwater under the same conditions.…”

GIPC: Glycosyl Inositol Phospho Ceramides, the major sphingolipids on earth

“…29 Finally, a phospholipase D activity that specifically hydrolyzes GIPCs and produces phytoceramide-1-phosphate (PC1P) have been recently identified in Brassicaceae. 30 Authors suggested that GIPC-PLD may play an important signaling role in the biological response in plant cells by producing signaling molecules, but the mechanism of regulation of the enzyme activity is largely unknown. GIPC-PLD could also play a role in de-anchoring GPIC-anchored proteins, see above.…”