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Isolation and Characterization of Sphingolipids in Arabidopsis Thaliana

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Isolation and Characterization of Sphingolipids in Arabidopsis Thaliana Isolation and Characterization of Sphingolipids in Arabidopsis thaliana A thesis submitted to the Miami University Honors Program in partial fulfillment of the requirements for University Honors by Marc Mecoli May, 2003 Oxford, Ohio ABSTRACT ISOLATION AND CHARACTERIZATION OF SPHINGOLIPIDS IN ARABIDOPSIS THALIANA by Marc Mecoli Total lipids were extracted from A. thaliana plant tissue and differential hydrolysis was utilized to determine the presence of sphingolipids. Silicic acid chromatography was utilized to separate lipids based upon their polarity, and fatty acid methyl esters were prepared from isolated A. thaliana sphingolipids. Gas chromatography and mass spectrometry were used to analyze the fatty acid content of the isolated sphingolipids. It was determined that the A. thaliana sphingolipids contained 16:0 and 18:0 fatty acids. The isolation and characterization scheme employed in this study will be used to investigate the sphingolipid fatty acid content of A. thaliana in the future. Isolation and Characterization of Sphingolipids in Arabidopsis thaliana by Marc Mecoli Approved by: _________________________, Advisor Brenda J. Blacklock _________________________, Advisor Ann E. Hagerman _________________________, Reader Michael W. Crowder Accepted by: _________________________, Director, University Honors Program Acknowledgements I thank Dr. Brenda Blacklock for all of her guidance and valuable input with this thesis. I would also like to thank Dr. Ann Hagerman and Dr. Michael Crowder for reading and commenting on my work. Finally, I thank my parents and my brothers, Greg and Jon, for being my greatest sources of inspiration and motivation. Table of Contents Item Page List of Figures iv List of Tables v Introduction 1 Methods and Materials 4 Growth, collection and storage of yeast and A. thaliana 4 Extraction and separation of lipids 5 Separation of sphingolipid standards via silicic acid chromatography 6 Differential hydrolysis of lipid standards 6 Differential hydrolysis of A. thaliana extract 6 Preparation of fatty acid methyl esters 8 A. thaliana FAME analysis via GC/MS 8 Results and Discussion 9 TLC plate stain selection 9 Biosil versus silicic acid chromatography 10 Separation of sphingolipid standards via silicic acid chromatography 12 Differential hydrolysis of lipid standards 13 Isolation of yeast and A. thaliana lipids 16 Differential hydrolysis of A. thaliana extract 17 A. thaliana FAME analysis via GC/MS 19 Conclusion 23 References 24 ii List of Figures Figure Page Figure 1. Representative structure of a sphingolipid 1 Figure 2. Diagram representing the sequential treatment of the extract during 7 the differential hydrolysis of A. thaliana Figure 3. TLC plates of yeast lipid extract separated by Biosil and silicic acid 11 column chromatography iii Figure 4. TLC plate of sphingolipid standard controls and separated 12 sphingolipid standards via silicic acid column chromatography. Figure 5. Representative diagrams of a triacylglycerol and a sphingolipid 13 Figure 6. TLC plate showing differential hydrolysis of lipid standards. 15 Figure 7. TLC plate of sphingolipid standard, yeast extract and A. thaliana 16 extract. Figure 8. TLC plate showing the differential hydrolysis of the glycolipid 18 fraction of A. thaliana extract. Figure 9 Reaction of an acyl lipid with MeOH which produces glycerol and 20 fatty acid methyl esters iv List of Tables Table Page Table 1. Rf values of galactocerebroside standard and suspected 17 sphingolipids from yeast and A. thaliana. Table 2. Mass spectral identification of methyl ester derivatives of 21 standard saturated fatty acids, standard glucocerebrosides, standard galactocerebrosides and A. thaliana sphingolipid fatty acids. v INTRODUCTION Sphingolipids are a class of lipids found in all eukaryotes and some bacteria and are important components of plant, animal and microorganism signal transduction systems and plasma membranes. In eukaryotic cells, sphingolipids are thought to increase the stability and decrease the permeability of membranes and have been implicated in regulating ion permeability (1). There is also growing evidence that sphingolipids are important in yeast cellular signaling (2). Clearly, the biosynthesis of sphingolipids is an integral part of a cell's life-sustaining function. The general sphingolipid structure contains three main components: a very long chain fatty acid (VLCFA), a long chain base and a head group (Figure 1). A characteristic amide linkage attaches the VLCFA to the long chain base. A ceramide, the simplest sphingolipid, consists of a long chain base, a very long chain fatty acid and hydroxy head group (Figure 1, R = OH). Cerebrosides such as galactocerebroside and glucocerebroside have a monosaccharide residue as the head group (Figure 1, R = OH long chain base R O NH OH C O very long chain fatty acid OH Figure 1. Representative structure of a sphingolipid having 4-hydroxy-8-sphingenine and 2-hydroxylignoceric (cerebronic) acid as the long chain base and fatty acid component, respectively. R represents a head group. 1 monosaccharide) (3). Our research laboratory is especially interested in the mechanisms by which the VLCFA moiety of sphingolipids is produced in plants. VLCFAs (fatty acids of 20 carbons and greater) are found in membrane sphingolipids, waxes, seed oil, and the extracellular pollen coat (1,4). The variety of chain lengths of VLCFAs and the diversity of lipids they are found in suggests that production of VLCFAs in plants is very complex. A better understanding of the metabolism of VLCFAs may provide us with genes for pharmaceutical and nutritional products as well as engineering plant products used for lubricants and fuel resources. VLCFAs are produced by the elongation of saturated and mono-unsaturated C18 fatty acids. Elongases (ELOs) are involved in the biosynthesis of VLCFAs in yeast. Studies have shown that disrupting expression of ELO genes in yeast reduces cellular levels of sphingolipid VLCFAs (5). However, little is known of the elongation of fatty acids to yield sphingolipid VLCFAs in plant systems. ELO homologues have been found by homology searches of the recently sequenced Arabidopsis thaliana genome; however, their role in sphingolipid biosynthesis is unclear. It is suspected that ELO homologues are involved in sphingolipid biosynthesis in the plant, particularly in the production of VLCFAs incorporated into sphingolipids. In our laboratory, the expression of several A. thaliana ELOs have been disrupted using techniques in molecular biology. However, no methods exist for isolating and characterizing the sphingolipid VLCFAs of resulting transgenic plants to determine the role of A. thaliana ELOs in sphingolipid biosynthesis. If the disrupted ELO is involved in sphingolipid VLCFA biosynthesis, then it is expected 2 that its disruption will significantly alter the biosynthesis of sphingolipids in the plant system. The purpose of this study was to develop methods for the isolation and characterization of sphingolipids in A. thaliana that will be used in future studies involving sphingolipid metabolism. Methods based on those developed for yeast and other plant systems were utilized to establish a scheme for the isolation of sphingolipids from A. thaliana (6-9). Total lipids were extracted from whole plant tissue and column chromatography was used to isolate polar lipids. Subjecting the polar lipids to mild alkaline conditions isolated sphingolipids by hydrolyzing the ester bond found in glycerolipids. After purification of the sphingolipid fraction, fatty acid methyl esters (FAMEs) were prepared and analyzed by GC/MS. Both 16:0 and 18:0 fatty acids were found in the isolated sphingolipid fraction. MATERIALS AND METHODS Growth, collection and storage of yeast and A. thaliana S. cerevisiae were grown in YPD media containing 1% yeast extract, 2% peptone and 2% dextrose. Cultures were grown for 17 h at 28 °C in a rotary shaker (300 cycles/min). Of this culture, 200 ml was transferred to 200 ml of complete minimal dropout media lacking uracil (cm-ura) supplemented with 0.34 g of yeast nitrogenous base (DIFCO), 1.0 g of (NH4)2SO4 and 2% dextrose per liter (10). This culture was grown for 48 h at 28 °C in a rotary shaker (300 cycles/min). Cells were harvested by centrifugation at 500 g (Centra-8, International Equipment Company) and immediately 3 treated with 5% trichloroacetic acid at 0 °C for 1.5 h to stop any phospholipase activity. The cell material was washed twice with distilled water, twice with 0.5% KH2PO4, resuspended in 0.5% KH2PO4, and then heated for 7 min at 100 °C to remove any residual trichloroacetic acid. The cell material was stored at -20 °C (11,12). A. thaliana plants were grown at 24 °C for 25 days with 16 h light / 8 h dark cycle. Freshly harvested A. thaliana stems, leaves and flowers were frozen on dry ice and homogenized with a liquid nitrogen-cooled mortar and pestle. The resulting powder was stored at -20 °C. Extraction and separation of lipids Lipids were extracted from yeast or 5 g of A. thaliana material with 2 ml of ethanol/water/ether/pyridine/NH4OH (15:15:5:1:0.018) at 60 °C (11). The solvent was evaporated under vacuum and the dried extract was dissolved in CHCl3 before storage at -20 °C. Silicic acid (100-mesh, Sigma) or Biosil (100-mesh, Biorad) column chromatography was used to separate the extracted material. The column system consisted of a 5.75-inch glass Pasteur pipette filled with approximately 2 ml of packed column material. The assembled column was washed with 5 ml of CHCl3 and 5 ml of acetone prior to loading a sample. Solvent was pushed through the column with a steady stream of N2. Lipid material was eluted with CHCl3 followed by acetone to separate neutral lipids and glycolipids, respectively. Each fraction was dried under N2, resuspended in CHCl3 and stored at -20 °C. 4 Extracted lipid material was applied to a TLC plate (Silica Gel, glass-backed, hard layer, fluorescent (GHFL), Analtech) and developed in CHCl3/CH3OH/NH4OH (66:17:3) (9). The plate was subjected to one of four staining systems (H2SO4/H2O (1:1), H2O/H2SO4/KCrO4 (27:20:3), 1% ceric ammonium nitrate in 20% H2SO4, or acetic acid/H2SO4/anisaldehyde (500:10:2)) (13,14).
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