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Title: SEPARATION AND IDENTIFICATION OF MAJOR PLANT SPHINGOLIPID CLASSES FROM LEAVES

Author
item MARKHAM, JONATHAN - D. DANFORTH PLANT SCI CTR
item LI, JIA - D. DANFORTH PLANT SCI CTR
item Cahoon, Edgar
item JAWORSKI, JAN - D. DANFORTH PLANT SCI CTR

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/9/2006
Publication Date: 8/11/2006
Citation: Markham, J., Li, J., Cahoon, E.B., Jaworski, J. 2006. Separation and identification of major plant sphingolipid classes from leaves. Journal of Biological Chemistry. 281:22684-22694.

Interpretive Summary: Sphingolipids provide structural integrity to the membranes of plant cells and contribute to the ability of crop plants to adapt to changes in environmental conditions, such as temperature and water status. By mediating the synthesis of sphingolipids, it may be possible to develop crops that are more resistant to environmental extremes. How sphingolipid synthesis is regulated in plants, however, is still largely unknown. Serine palmitoyltransferase (SPT), the enzyme that catalyzes the first step in sphingolipid synthesis, likely plays an important role in regulating this pathway. In this study, the properties of a bacterial SPT were examined to provide insights into the function of the plant SPT. The bacterial SPT, unlike the plant enzyme, can be produced and purified in large quantities in a recombinant form, which facilitates detailed analyses of this enzyme. The results from these studies provided evidence that supports structural models for the active site of SPT in plants and other eukaryotes. This research will be useful for plant biochemists and molecular biologists who are attempting to improve the environmental adaptability of crops. This research will ultimately benefit the U.S. farmer by aiding in the development of higher yielding and higher value crops.

Technical Abstract: 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, a method for the separation of sphingolipid classes from A. thaliana leaves is described that uses the hydrolysis of sphingolipids and HPLC analysis of o-phthaldialdehyde derivatives of released long-chain bases to monitor 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-inositolphospho-ceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. In extracts from A. thaliana leaves, hexose-hexuronic-inositolphosphoceramides, monohexosylceramides and ceramides accounted for approximately 64%, 34% and 2% of the total sphingolipids, respectively. The methods described were used to extract and separate the sphingolipids from soybean and tomato. Like A. thaliana the neutral sphingolipids consisted of ceramide and monohexosylceramides, however, the major polar sphingolipid from tomato and soybean was found to be, as previously identified, N-acetyl-hexosamine-hexuronic-inositol-phosphoceramide. The data indicate the general applicability of these methods for analysis of total sphingolipids from plants.