Plant lipidomics: Refining pathway knowledge with a new technology

The large majority the knowledge we have on plant metabolism was generated before liquid chromatography-mass spectrometry-based (LC-MS) lipidomics was available. This translates into the fact that we have a new technique which results can confirm or not reviously generated hypotheses. Interestingly, some of our observations on lipid composition in the plant model Arabidopsis thaliana showed discrepancies with generally accepted ideas. we reported the existence of new compounds belonging to ER-synthesized classes of lipids containing 16:3 or its precursor 16:21, fatty acids that are typical of the chloroplast and deem to be absent in lipids with ER origin. The challenge imposed by these compounds was big: how to explain the existence of compounds that are not supposed to exist, and more importantly, how to reconcile it with more than 30 years of studies in lipid biochemistry.

In 1982, Roughan and Slack2 published one of the most influential reviews in the field of plant lipids. They compiled and systematized the evidence on lipid synthesis in the chloroplast and ER of plant leaf cells. They compared radioactive labeling studies on isolated chloroplast with studies on full leaves. I will make my best attempt to summarize their conclusions while at the time provide proper background for the subject. Chloroplast can use acetate very effectively as a substrate for fatty acid synthesis. When fed with radioactive acetate, chloroplasts incorporate radioactivity very quickly into the fatty acids 16:0 and 18:1, which means they are the most abundant substrates for glycerolipid assembly. Interestingly, chloroplast and ER have different preferences for assembling these fatty acids. The chloroplast synthesizes a sn-1-18:1-sn-2-16:0 phosphatidic acid (PA) whereas the ER synthesizes either a sn-1-16:0 -sn-2-18:1 or a sn-1-18:1-sn-2-18:1 configuration, determining at this step the stereochemistry of the lipids synthesized at both compartments. From that point on 16:0 and 18:1 may undergo the following desaturation pathways: 16:0 → 16:1 → 16:2 → 16:3 and 18:1 → 18:2 → 18:3, respectively. In those labeling experiments, the desaturation of 16:0 was only observed in the chloroplast, and only for the lipid MGDG. The label of 16:0 would remain high during the whole duration of labeling experiments for all the other compounds4. Radiolabeling studies were largely in agreement with Arabidopsis fatty acid composition3, in which except for DGDG, the metabolic child of MGDG, the levels of 16:1, 16:2 and 16:3 in other lipids were either undetected or neglectable.

These studies, together with the vast majority of works in the field of plant lipids relied on the technique of thin-layer chromatography (TLC) coupled to gas chromatography-flame ion detection (GC-FID). With these methods, fatty acids are excised from lipid molecules and measured prior derivatization by GC-FID. In contrast, measurements by LC-MS can measure whole lipid molecules, which can potentially help understand better the different pools of compounds that exist in lipid metabolism. Fatty acid composition can be then acquired by ion fragmentation of the molecules. As we mentioned before, we found compounds that did not match the possible fatty acid composition for ER or chloroplast compounds. These compounds are 34:6 PC, 34:6 PE, 32:3 PI, 34:5 PI and 34:6 SQDG. According to fragmentation data they contained either 16:3 or its precursor 16:2: (16:3/18:3) PC, (16:3/18:3 PE), (16:0/18:3) PI, (18:3/16:2) PI and (18:3/16:3) SQDG2. Interestingly, a pioneer study of plant lipidomics in Ruth Welti’s group4 reported as well a few compounds that did not follow the paradigm set by labeling studies, although they did not discuss the findings. They found (16:2/18:3) SQDG, (16:3/18:3) SQDG, (16:1/18:3) PE, (16:1/18:3) PC, (16:1/18:3) PI, and (16:1/18:3) PS. These compounds and the ones we detected are all low in abundance. It is possible that the high sensitivity of liquid chromatography allowed us to detect them. In a recent study5, we compared the sensitivity of ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) with that of gas chromatography with flame ionization detector (GC-FID), traditionally used to measure fatty acids. We hydrolyzed fatty acids from lipid extracts of Arabidopsis leaves, to make a fair comparison between the two techniques. We could detect 20 different fatty acids with UPLC-MS, whereas 8 fatty acids were detected with GC-FID.

We proposed how to explain the newly found compounds considering the existent theory. The low level of lipids containing unsaturated 16C, suggest is in part an issue of sensitivity of the previous approaches. The presence of unsaturated 16C could be possible due to a residual activity of the enzyme SSI2 for 16:06. This enzyme has a high substrate preference of the soluble desaturase SSI2 for 18:0 versus 16:0, and it is responsible for the high unsaturation level of 18C chains in glycerolipids, while 16C chains are kept as 16:0. The processing of 16:1 chains by also residual activities of thioesterases FATA and FATB could make available 16:1 to the ER7.

We are currently studying unsaturated 16C from the ER back into the chloroplast. In order to do this, we adapted Rhyzopus lipase digestion to UPLC fractions. Rhyzopus lipase digestion determines whether the 16C chain is at the sn-1 and sn-2 position of glycerolipids. This way we are able to determine how much of each detected species of chloroplast lipids comes from the chloroplast itself and how much from the endoplasmic reticulum. This would not only shed light into the pathway of unsaturated 16C. Providing information into the origin of lipid species detected by lipidomics will help further lipidomic studies to do a more insightful interpretation of data. (Studied by Dr. Asdrubal Burgos and Urszula Luzarowska

References

1 Burgos, A., Szymanski, J., Seiwert, B., Degenkolbe, T., Hannah, M.A., Giavalisco, P. and Willmitzer, L. (2011) Analysis of short-term changes in the Arabidopsis thaliana glycerolipidome in response to temperature and light. Plant Journal, 656-668.

2 Roughan PG, Slack CR. Cellular-Organization of Glycerolipid Metabolism. Annu Rev Plant Phys 1982; 33: 97-132.

3 Siebertz, H.P. and Heinz, E. (1977) Labelling experiments on the origin of hexa- and octa-decatrienoic acis in galactolipids from leaves. Z. Naturforsch. , 32c, 193-205.

4 Devaiah, S.P., Roth, M.R., Baughman, E., Li, M., Tamura, P., Jeannotte, R., Welti, R. and Wang, X. (2006) Quantitative profiling of polar glycerolipid species from organs of wild-type Arabidopsis and a phospholipase D-alpha1 knockout mutant. Phytochemistry, 67, 1907-1924.

5 Bromke, M.A., Hochmuth, A., Tohge, T., Fernie, A.R., Giavalisco, P., Burgos, A., Willmitzer, L., Brotman, Y. (2015) Liquid chromatography high-resolution mass spectrometry for fatty acid profiling. Plant Journal, 81(3), 529-536

6 Kachroo, A., Shanklin, J., Whittle, E., Lapchyk, L., Hildebrand, D. and Kachroo, P. (2007) The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis. Plant Molecular Biology, 63, 257-271.

7 Salas, J.J. and Ohlrogge, J.B. (2002) Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases. Archives of Biochemistry and Biophysics, 403, 25-34.

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