Method for isolation and quantification of inositol glycan produced by glycosylinositol phosphoceramide-hydrolyzing phospholipase D in plants

Glycosylinositol phosphoceramide (GIPC) is the most abundant sphingolipids in plants. Previously, we found phospholipase D (PLD) activity that hydrolyzes GIPC to phytoceramide 1-phosphate (PCerP) in plants and revealed that GIPC-PLD activity is carried out by an enzyme encoded by non-specific phospholipase C3 (NPC3) gene. In this study, we established a method for isolation and quantification of inositol glycan (InoGly), a counterpart of PCerP produced from GIPC, using TLC imaging. We confirmed that A. thaliana NPC3 protein and partially purified GIPC-PLD from cabbage produced InoGly in a similar amount to that of PCerP from purified GIPC. We applied our method to determination of InoGly present in plant tissues and found that it was present at 40-80 nmol/g (wet wt.) in cabbage leaves, radish root and broccoli stem, and increased to 80-120 nmol/g after homogenization of the tissues. Similar increases in PCerP and decreases in GIPC were observed after homogenization, indicating that InoGly and PCerP were produced from GIPC by GIPC-PLD activity in response to homogenization. We believe our method, which does not require the complicated process or large device, will contribute to a better understanding of GIPC metabolism and signaling in plants.