Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for ϳ64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.Sphingolipids are recognized as universal components of eukaryotic membranes with a diverse array of functions (1-3). Recent interest in sphingolipids from plants has been stimulated by the realization that they may form a significant proportion of the plasma membrane (4), potentially as lipid rafts (5, 6), are involved in signaling a plant's response to drought (7,8), and regulate the ultimate fate of plant cells through programmed cell death (9, 10). In order to understand the role of plant sphingolipids in this diverse array of already discovered roles and to determine what other biological functions sphingolipids may have in plants, it is necessary to be able to measure the sphingolipid content in a qualitative and quantitative way (11). Sphingolipid signaling is thought to be a complex multifactorial signal derived from the interaction of several different sphingolipids (12), making the examination of all sphingolipids a critical factor in the dissection of sphingolipid function. Thus, the emerging field of sphingolipidomics has received much attention in animal biology but remains neglected in plants (13).Previous studies on plant sphingolipids have exclusively concentrated...
