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J. Biol. Chem., Vol. 281, Issue 32, 22684-22694, August 11, 2006

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Separation and Identification of Major Plant Sphingolipid Classes from Leaves^*

Jonathan E. Markham¹, Jia Li, Edgar B. Cahoon, and Jan G. Jaworski

From the Donald Danforth Plant Science Center and United States Department of Agriculture-Agricultural Research Service Plant Genetics Research Unit, Saint Louis, Missouri 63132

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 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.

Received for publication, April 27, 2006 , and in revised form, June 9, 2006.

^* This work was supported by National Science Foundation 2010 Grant 0312559. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Donald Danforth Plant Science Center, 975 N. Warson Rd., Saint Louis, MO 63132. Tel.: 314-587-1644; Fax: 314-587-1744; E-mail: jmarkham{at}danforthcenter.org.

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