Abstract
Lipids are produced through a dynamic metabolic network involving branch points, cycles, reversible reactions, parallel reactions in different subcellular compartments, and distinct pools of the same lipid class involved in different parts of the network. For example, diacylglycerol (DAG) is a biosynthetic and catabolic intermediate of many different lipid classes. Triacylglycerol can be synthesized from DAG assembled de novo, or from DAG produced by catabolism of membrane lipids, most commonly phosphatidylcholine. Quantification of lipids provides a snapshot of the lipid abundance at the time they were extracted from the given tissue. However, quantification alone does not provide information on the path of carbon flux through the metabolic network to synthesize each lipid. Understanding lipid metabolic flux requires tracing lipid metabolism with isotopically labeled substrates over time in living tissue. [14C]acetate and [14C]glycerol are commonly utilized substrates to measure the flux of nascent fatty acids and glycerol backbones through the lipid metabolic network in vivo. When combined with mutant or transgenic plants, tracing of lipid metabolism can provide information on the molecular control of lipid metabolic flux. This chapter provides a method for tracing in vivo lipid metabolism in developing Arabidopsis thaliana seeds, including analysis of 14C labeled lipid classes and fatty acid regiochemistry through both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) approaches.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Roughan PG, Slack CR (1982) Cellular-organization of glycerolipid metabolism. Annu Rev Plant Physiol Plant Mol Biol 33:97–132. https://doi.org/10.1146/annurev.pp.33.060182.000525
Browse J, Somerville C (1991) Glycerolipid synthesis—biochemistry and regulation. Annu Rev Plant Physiol Plant Mol Biol 42:467–506. https://doi.org/10.1146/annurev.pp.42.060191.002343
Harwood JL (1996) Recent advances in the biosynthesis of plant fatty acids. Biochim Biophys Acta 1301(1–2):7–56. https://doi.org/10.1016/0005-2760(95)00242-1
Bates PD, Browse J (2012) The significance of different diacylglycerol synthesis pathways on plant oil composition and bioengineering. Front Plant Sci 3:147. https://doi.org/10.3389/fpls.2012.00147
Allen DK, Bates PD, Tjellström H (2015) Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: past, present and future. Progr Lipid Res 58:97–120. https://doi.org/10.1016/j.plipres.2015.02.002
Bates PD (2016) Understanding the control of acyl flux through the lipid metabolic network of plant oil biosynthesis. Biochim Biophys Acta 1861(9, Part B):1214–1225. https://doi.org/10.1016/j.bbalip.2016.03.021
Kunst L, Browse J, Somerville C (1988) Altered regulation of lipid biosynthesis in a mutant of Arabidopsis deficient in chloroplast glycerol-3-phosphate acyltransferase activity. Proc Natl Acad Sci U S A 85(12):4143–4147. https://doi.org/10.1073/pnas.85.12.4143
Xu CC, Fan JL, Riekhof W, Froehlich JE, Benning C (2003) A permease-like protein involved in ER to thylakoid lipid transfer in Arabidopsis. EMBO J 22(10):2370–2379. https://doi.org/10.1093/emboj/cdg234
Bates PD, Browse J (2011) The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. Plant J 68(3):387–399. https://doi.org/10.1111/j.1365-313X.2011.04693.x
Bates PD, Fatihi A, Snapp AR, Carlsson AS, Browse J, Lu C (2012) Acyl editing and headgroup exchange are the major mechanisms that direct polyunsaturated fatty acid flux into triacylglycerols. Plant Physiol 160(3):1530–1539. https://doi.org/10.1104/pp.112.204438
Bates PD, Johnson SR, Cao X, Li J, Nam J-W, Jaworski JG, Ohlrogge JB, Browse J (2014) Fatty acid synthesis is inhibited by inefficient utilization of unusual fatty acids for glycerolipid assembly. Proc Natl Acad Sci U S A 111(3):1204–1209. https://doi.org/10.1073/pnas.1318511111
Karki N, Johnson BS, Bates PD (2019) Metabolically distinct pools of phosphatidylcholine are involved in trafficking of fatty acids out of and into the chloroplast for membrane production. Plant Cell 31(11):2768–2788. https://doi.org/10.1105/tpc.19.00121
Tjellström H, Strawsine M, Ohlrogge JB (2015) Tracking synthesis and turnover of triacylglycerol in leaves. J Exp Bot 66(5):1453–1461. https://doi.org/10.1093/jxb/eru500
Focks N, Benning C (1998) wrinkled1: a novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism. Plant Physiol 118(1):91–101. https://doi.org/10.1104/pp.118.1.91
Baud S, Boutin JP, Miquel M, Lepiniec L, Rochat C (2002) An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol Biochem 40(2):151–160. https://doi.org/10.1016/S0981-9428(01)01350-X
Li YH, Beisson F, Pollard M, Ohlrogge J (2006) Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation. Phytochemistry 67(9):904–915. https://doi.org/10.1016/j.phytochem.2006.02.015
Karki N, Bates PD (2018) The effect of light conditions on interpreting oil composition engineering in Arabidopsis seeds. Plant Direct 2(6):e00067. https://doi.org/10.1002/pld3.67
Bao XM, Pollard M, Ohlrogge J (1998) The biosynthesis of erucic acid in developing embryos of Brassica rapa. Plant Physiol 118(1):183–190. https://doi.org/10.1104/pp.118.1.183
Shintani DK, Ohlrogge JB (1995) Feedback inhibition of fatty-acid synthesis in tobacco suspension cells. Plant J 7(4):577–587. https://doi.org/10.1046/j.1365-313X.1995.7040577.x
Bates PD, Ohlrogge JB, Pollard M (2007) Incorporation of newly synthesized fatty acids into cytosolic glycerolipids in pea leaves occurs via acyl editing. J Biol Chem 282(43):31206–31216. https://doi.org/10.1074/jbc.M705447200
Bates PD, Durrett TP, Ohlrogge JB, Pollard M (2009) Analysis of acyl fluxes through multiple pathways of triacylglycerol synthesis in developing soybean embryos. Plant Physiol 150(1):55–72. https://doi.org/10.1104/pp.109.137737
Tjellström H, Yang Z, Allen DK, Ohlrogge JB (2012) Rapid kinetic labeling of Arabidopsis cell suspension cultures: implications for models of lipid export from plastids. Plant Physiol 158(2):601–611. https://doi.org/10.1104/pp.111.186122
Zhou XR, Bhandari S, Johnson BS, Kotapati HK, Allen DK, Vanhercke T, Bates PD (2020) Reorganization of acyl flux through the lipid metabolic network in oil-accumulating tobacco leaves. Plant Physiol 182(2):739–755. https://doi.org/10.1104/pp.19.00667
Lu C, Xin Z, Ren Z, Miquel M, Browse J (2009) An enzyme regulating triacylglycerol composition is encoded by the ROD1 gene of Arabidopsis. Proc Natl Acad Sci U S A 106(44):18837–18842. https://doi.org/10.1073/pnas.0908848106
Yang W, Wang G, Li J, Bates PD, Wang X, Allen DK (2017) Phospholipase Dζ enhances diacylglycerol flux into triacylglycerol. Plant Physiol 174(1):110–123. https://doi.org/10.1104/pp.17.00026
Williams JP, Imperial V, Khan MU, Hodson JN (2000) The role of phosphatidylcholine in fatty acid exchange and desaturation in Brassica napus L. leaves. Biochem J 349:127–133. https://doi.org/10.1042/0264-6021:3490127
Slack CR, Roughan PG, Balasingham N (1977) Labeling studies in vivo on metabolism of acyl and glycerol moieties of glycerolipids in developing maize leaf. Biochem J 162(2):289–296. https://doi.org/10.1042/bj1620289
Bates PD, Browse J (2011) The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds. Plant J 68(3):387–399. https://doi.org/10.1111/j.1365-313X.2011.04693.x
Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem Soc Trans 11(5):591–592. https://doi.org/10.1042/bst0110591
Li-Beisson Y, Shorrosh B, Beisson F, Andersson MX, Arondel V, Bates PD, Baud S, Bird D, Debono A, Durrett TP, Franke RB, Graham IA, Katayama K, Kelly AA, Larson T, Markham JE, Miquel M, Molina I, Nishida I, Rowland O, Samuels L, Schmid KM, Wada H, Welti R, Xu C, Zallot R, Ohlrogge J (2013) Acyl-lipid metabolism. Am Soc Plant Biol 11:e0161. https://doi.org/10.1199/tab.0161
Lin JT (2007) HPLC separation of acyl lipid classes. J Liquid Chromatogr Rel Technol 30(14):2005–2020. https://doi.org/10.1080/10826070701435020
Kotapati HK, Bates PD (2018) A normal phase high performance liquid chromatography method for the separation of hydroxy and non-hydroxy neutral lipid classes compatible with ultraviolet and in-line liquid scintillation detection of radioisotopes. J Chromatogr B 1102-1103:52–59. https://doi.org/10.1016/j.jchromb.2018.10.012
Kotapati HK, Bates PD (2020) Normal phase HPLC method for combined separation of both polar and neutral lipid classes with application to lipid metabolic flux. J Chromatogr B 1145:122099. https://doi.org/10.1016/j.jchromb.2020.122099
Cahoon EB, Dietrich CR, Meyer K, Damude HG, Dyer JM, Kinney AJ (2006) Conjugated fatty acids accumulate to high levels in phospholipids of metabolically engineered soybean and Arabidopsis seeds. Phytochemistry 67(12):1166–1176. https://doi.org/10.1016/j.phytochem.2006.04.013
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kotapati, H.K., Bates, P.D. (2021). 14C-Tracing of Lipid Metabolism. In: Bartels, D., Dörmann, P. (eds) Plant Lipids. Methods in Molecular Biology, vol 2295. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1362-7_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1362-7_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1361-0
Online ISBN: 978-1-0716-1362-7
eBook Packages: Springer Protocols