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

<jats:p>The structure and biosynthetic route for an unidentified lipid (lipid X) detected by <jats:styled-content style="fixed-case">TLC</jats:styled-content> of cabbage (<jats:italic>Brassica oleracea</jats:italic>) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by <jats:styled-content style="fixed-case">MALDI</jats:styled-content>‐<jats:styled-content style="fixed-case">TOF MS</jats:styled-content> 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 <jats:styled-content style="fixed-case">GC</jats:styled-content>‐<jats:styled-content style="fixed-case">MS</jats:styled-content> 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 <jats:styled-content style="fixed-case">MALDI</jats:styled-content>‐<jats:styled-content style="fixed-case">TOF MS</jats:styled-content> with Phos‐tag. From these results, lipid X was determined to be phytoceramide‐1‐phosphate (<jats:styled-content style="fixed-case">PC</jats:styled-content>1P) with an α‐hydroxy fatty acid. When cabbage homogenates were incubated, <jats:styled-content style="fixed-case">PC</jats:styled-content>1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide (<jats:styled-content style="fixed-case">GIPC</jats:styled-content>). The formation of <jats:styled-content style="fixed-case">PC</jats:styled-content>1P from <jats:styled-content style="fixed-case">GIPC</jats:styled-content> was confirmed by treatment of purified cabbage <jats:styled-content style="fixed-case">GIPC</jats:styled-content> with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes <jats:styled-content style="fixed-case">GIPC</jats:styled-content> specifically, but not glycerophospholipids and sphingomyelin. <jats:italic>Arabidopsis thaliana</jats:italic> also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes <jats:styled-content style="fixed-case">GIPC</jats:styled-content> produces <jats:styled-content style="fixed-case">PC</jats:styled-content>1P in brassicaceous plants.</jats:p>