Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.
Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.
TLC coupled with GLC provides a robust and rapid tool for quantitative analysis of polar lipids in plants. Small changes in lipid composition can be identified; therefore, this method has been used for large scale screening of mutants impaired in polar lipid metabolic pathways1,20. This method is also widely used for monitoring activities of enzymes utilizing polar lipids as substrate.2,6,7
Besides leaves, the lipid composition of other plant tissues such as roots and seeds or subcellular fractions such as chloroplasts and mitochondria can also be determined in the same way.
The solvent system (acetone, toluene, water) used here is optimized for the separation of glycolipids and phospholipids in plants. However, in tgd1,2,3,4 mutants and isolated chloroplasts, TGDG runs together with PE while tetragalactosyldiacylglycerol runs with PC. In this case a solvent system with chloroform, methanol, acetic acid and water (85: 20: 10: 4, v/v/v/v) is used13. Sometimes two-dimensional TLC using two different solvent systems is performed to further separate glycolipids and phospholipids19. In addition, plant tissues can be directly subjected to the FAME reaction followed by GLC to determine the total fatty acid profile without initial separation on TLC5. Beside the demonstrated TLC-GLC system, another method used for lipid profiling is based on direct electrospray ionization tandem mass spectrometry17. In this method the initial chromatographic separation of lipids in the extract is omitted. However, this method requires expensive equipment and experienced personnel, which makes it less useful for routine analyses in the lab or for mutant screening.
The authors have nothing to disclose.
This work is supported by a grant from US National Science Foundation to Christoph Benning.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
α-naphthol | Sigma-Aldrich | N1000 | ||
Methanolic HCL 3N | Sigma-Aldrich | 33050-U | Dilute to 1N by methanol | |
Si250-PA TLC plates | J.T.Baker | 7003-04 | With pre-absorbent | |
TLC chamber | Sigma-Aldrich | Z266000 | ||
Screw cap tubes | VWR | 53283-800 | ||
Scew caps | Sun Sri | 13-425 | ||
PTFE disk | Sun Sri | 200 608 | ||
GLC system | Hewlett Packard | HP6890 | ||
DB-23 column | J&W Scientific | 122-2332 | ||
GLC vials | Sun Sri | 500 132 | ||
Caps of GLC vials | Sun Sri | 201 828 | ||
Chemstation software | Agilent | G2070AA |