Department of Biochemistry and Molecular Biology, Michigan State University
Wang, Z., Benning, C. Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC). J. Vis. Exp. (49), e2518, doi:10.3791/2518 (2011).
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.
1. Lipid Extraction
2. Thin Layer Chromatography (TLC)15
3. Fatty Acyl Methylester (FAME) Reaction16
4. Gas-Liquid Chromatography (GLC)10
5. Representative Results:
Examples of irreversible staining of TLC-separated lipids from 4-week-old Arabidopsis seedlings are shown in Figure 2. The sulfuric acid stained lipids (Figure 2A) are charred and appear as brown spots. α-naphthol is preferred to stain glycolipids such as MGDG, DGDG, SQDG etc. Glycolipids stained with α-naphthol carry a pink-purple color while other polar lipids stain yellow (Figure 2B). The iodine staining is reversible and gives lipids a yellowish color that will disappear over a short time as iodine evaporates (Figure 2C). Briefly iodine stained lipids can be subjected to GLC analysis although unstained lipids are preferable to reduce break down of lipids.
If done successfully, distinctive signals representing different Fatty acyl methylester will be observed after GLC (Figure 3). Fatty acyl methylester with shorter carbon chain and fewer double bonds have shorter retention time using the DB-23 column. Fatty acyl methylester profiling is a sensitive tool to identify mutants with altered lipid composition. In Figure 4, the MGDG18:3 fatty acid molar ratio is decreased in the tgd4-1 mutant compared to the wild type18. By dividing the moles of Fatty acyl methylester for one lipid class with the moles of all lipid classes, the molar ratio of each lipid are calculated. For example, to calculate the molar ratio of MGDG:
(MGDG) mol% = ∑ [FAMEs(MGDG)] / ∑ [FAMEs(total)] x100%
The resulting molar ratios of each lipid class from both the wild type and the mutant can be compared. For instance, the tgd4-1 mutant has increased relative amounts of MGDG and PG but decreased amounts of DGDG and PE (Figure 5)18.
Figure 1. Flow chart of polar lipid analysis using Arabidopsis seedlings. Total lipids are extracted from 4-week-old Arabidopsis seedlings and separated by TLC. The separated lipids can be scraped from TLC plate for transesterification followed by GLC analysis.
Figure 2. Separation of lipids on TLC plates. Lipid extracts of 35 mg (fresh weight) wild type seedlings are separated by TLC and stained by sulfuric acid (A), α-naphthol (B) or iodine vapor (C). Three repeats are shown in each staining method. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; SQDG, sulfoquinovosyldiacylglycerol.
Figure 3. GLC analysis of Fatty Acid Methylesters (FAMEs) derived from MGDG of the wild type. FAMEs are separated on a 30 m capillary column and detected by flame ionization. Pentadecanoic acid (15:0) is used as an internal standard.
Figure 4. Fatty acid profile of MGDG in the wild type Col2 (white columns) and the tgd4-1 mutant (black columns). Fatty acids are presented as the number of carbons followed by the number of double bonds. Three repeats are averaged and standard deviations are shown.
Figure 5. Polar lipids composition of the wild type Col2 (white columns) and the tgd4-1 mutant (black columns). Three repeats are averaged and standard deviations are shown by error bar.
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.
No conflicts of interest declared.
This work is supported by a grant from US National Science Foundation to Christoph Benning.
|Methanolic HCL 3N||Sigma-Aldrich||33050-U||Dilute to 1N by methanol|
|Si250-PA TLC plates||JT Baker||7003-04||With pre-absorbent|
|Screw cap tubes||VWR international||53283-800|
|Scew caps||Sun Sri||13-425|
|PTFE disk||Sun Sri||200 608|
|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 Technologies||G2070AA|