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Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves

用14C -乙酸酯脉冲追逐标记法研究植物叶片中脂肪酸和甘油脂代谢

LY Linhui Yu
CZ Chao Zhou
JF Jilian Fan
CX Changcheng Xu

发布: 2021年02月05日第11卷第3期 DOI: 10.21769/BioProtoc.3900 浏览次数: 4345

评审: Agnieszka ZienkiewiczAnonymous reviewer(s)

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Cover of The Plant Cell, featuring study using the protocol.

Jul 2019

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Abstract

Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. The basic principle of isotope labeling and its recent technological advances have been reviewed (Allen et al., 2015). The present protocol describes the use of 14C-labeled acetate to determine rates of fatty acid synthesis and degradation and to track the metabolism of glycerolipids in leaves. This method, which is often referred to as acetate pulse-chase labeling, has been widely used to probe various aspects of lipid metabolism (Allen et al., 2015), including the role of autophagy in membrane lipid turnover (Fan et al., 2019) and the interplay between lipid and starch metabolism pathways (Yu et al., 2018).

Keywords: 14C-acetate pulse-chase (碳14乙酸酯脉冲追踪) search Fatty acid (脂肪酸) search Lipid (脂质) search

Materials and Reagents

  1. Pipette tips

  2. 90 mm Petri dish (Thermo Fisher Scientific, catalog number: 101R20 )

  3. 1.5 ml and 0.5 ml Eppendorf safe-lock microcentrifuge tube

  4. TLC Silica gel 60 plate (Merck)

  5. Filter paper

  6. Pencil

  7. 3-4-week-old Arabidopsis plants

  8. 14C-acetate (American Radiolabeled Chemicals, catalog number: ARC 0101D-1 mCi )

  9. Double-distilled water (ddH2O)

  10. MES (Sigma-Aldrich, catalog number: M3671 )

  11. MS salt (Gentaur, catalog number: LSP03-50LT )

  12. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )

  13. Chloroform (Thermo Fisher Scientific, catalog number: C607 )

  14. Methanol (VWR Chemicals, catalog number: BDH1135-4LG )

  15. Acetic acid (Macron, catalog number: MK-8817-500 )

  16. Acetone (Macron, catalog number: MK244310 )

  17. Toluene (VWR Chemicals, catalog number: BDH1151-4LG )

  18. Hexane (VWR Chemicals, catalog number: BDH1129-4LG )

  19. Diethyl ether (Macron, catalog number: MK085010 )

  20. (NH4)2SO4 (Mallinckrodt Chemicals, catalog number: 3512-12 )

  21. Nitrogen gas

  22. Scintillation liquid (PerkinElmer, catalog number: 6013199 )

  23. Tween-20 (Sigma-Aldrich, CAS number: 9005-64-5 )

  24. H3PO4 (Sigma-Aldrich, CAS number: 7664-38-2 )

  25. Labeling medium (see Recipes)

  26. Lipid extraction solution (see Recipes)

  27. KCl solution (see Recipes)

  28. Developing solvent 1 (see Recipes)

  29. Developing solvent 2 (see Recipes)

Equipment

  1. Pipettes

  2. Analytical balance (Mettler Toledo, model: AE100 )

  3. Shaker (Bellco Biotechnology, model: 7644-10115 )

  4. Vortexer (Denville Scientific, model: S7030 )

  5. Fume hood

  6. Nitrogen evaporator (Thermo Fisher Scientific, model: TS-18825 )

  7. Oven (Thermo Fisher Scientific, model: 13-247-650G )

  8. Personal safety equipment: lab coat, nitrile gloves, and eye protection

  9. Cork borer

  10. TLC developing tank

  11. Scissors

  12. Survey Meter (Ludlum Measurements, model: Model 3 )

  13. Autoradiography cassette (FUJIFILM, model: BAS 2040 )

  14. Phosphor screen (FUJIFILM, model: BAS-SR-2040 )

  15. Scintillation analyzer (PerkinElmer, model: Tri-Carb 2900TR )

  16. Typhoon FLA 7000 biomolecular imager (GE Healthcare)

  17. -80 °C and -20 °C freezer

  18. Centrifuge (Eppendorf, model: 5425 )

Software

  1. ImageQuant5.2 (GE Healthcare)

Procedure

English
中文翻译

文章信息

版权信息

© 2021 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Yu, L., Zhou, C., Fan, J. and Xu, C. (2021). Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves. Bio-protocol 11(3): e3900. DOI: 10.21769/BioProtoc.3900.

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分类

生物化学 > 脂质

植物科学 > 植物生物化学 > 代谢物

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