Acyl-Lipid Metabolism Author(s): Yonghua Li-Beisson , Basil Shorrosh , Fred Beisson , Mats X. Andersson , Vincent Arondel , Philip D. Bates , Sébastien Baud , David Bird , Allan DeBono , Timothy P. Durrett , Rochus B. Franke , Ian A. Graham , Kenta Katayama , Amélie A. Kelly , Tony Larson , Jonathan E. Markham , Martine Miquel , Isabel Molina , Ikuo Nishida , Owen Rowland , Lacey Samuels , Katherine M. Schmid , Hajime Wada , Ruth Welti , Changcheng Xu , Rémi Zallot , and John Ohlrogge Source: The Arabidopsis Book, Number 11 2013. Published By: The American Society of Plant Biologists URL: http://www.bioone.org/doi/full/10.1199/tab.0161 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. The Arabidopsis Book © 2013 American Society of Plant Biologists First published on January 29, 2013: e0161. doi: 10.1199/tab.0161 This chapter is an updated version of a chapter originally published on June 11, 2010, e0133. doi: 10.1199/tab.0133 Acyl-Lipid Metabolism Yonghua Li-Beisson,* Basil Shorrosh, Fred Beisson, Mats X. Andersson, Vincent Arondel, Philip D. Bates, Sébastien Baud, David Bird, Allan DeBono, Timothy P. Durrett, Rochus B. Franke, Ian A. Graham, Kenta Katayama, Amélie A. Kelly, Tony Larson, Jonathan E. Markham, Martine Miquel, Isabel Molina, Ikuo Nishida, Owen Rowland, Lacey Samuels, Katherine M. Schmid, Hajime Wada, Ruth Welti, Changcheng Xu, Rémi Zallot, and John Ohlrogge *Correspondence to [email protected] Acyl lipids in Arabidopsis and all other plants have a myriad of diverse functions. These include providing the core diffu- sion barrier of the membranes that separates cells and subcellular organelles. This function alone involves more than 10 membrane lipid classes, including the phospholipids, galactolipids, and sphingolipids, and within each class the variations in acyl chain composition expand the number of structures to several hundred possible molecular species. Acyl lipids in the form of triacylglycerol account for 35% of the weight of Arabidopsis seeds and represent their major form of carbon and energy storage. A layer of cutin and cuticular waxes that restricts the loss of water and provides protection from invasions by pathogens and other stresses covers the entire aerial surface of Arabidopsis. Similar functions are provided by suberin and its associated waxes that are localized in roots, seed coats, and abscission zones and are produced in response to wounding. This chapter focuses on the metabolic pathways that are associated with the biosynthesis and degradation of the acyl lipids mentioned above. These pathways, enzymes, and genes are also presented in detail in an associated website (ARALIP: http:// aralip.plantbiology.msu.edu/). Protocols and methods used for analysis of Arabidopsis lipids are provided. Finally, a detailed summary of the composition of Arabidopsis lipids is provided in three figures and 15 tables. CONTENTS 3. METHODS AND PROTOCOLS FOR ARABIDOPSIS LIPID ANALYSES ......................................................................... 30 1. INTRODUCTION ................................................................... 2 3.1. Lipid Extraction Methods and Separation ................... 30 3.2. Determination of Total Fatty Acid Profiles ................... 32 2. SUMMARY AND PERSPECTIVES ON MAJOR PATHWAYS OF ACYL-LIPID METABOLISM IN ARABIDOPSIS ............. 3 3.3. Glycerolipid Analysis Methods .................................... 33 3.4. Seed Oil Quantification ............................................... 35 2.1. Fatty Acid Synthesis and Export ..................................... 3 3.5. TAG Analysis by Liquid Chromatography 2.2. Plastid Glycerolipid Synthesis......................................... 5 Mass Spectrometry .................................................... 35 2.3. Eukaryotic Phospholipid Synthesis ................................. 7 3.6. Acyl-CoA Analysis by High-Performance Liquid Chromatography .............................................. 37 2.4. Sphingolipid Synthesis ................................................. 10 3.7. Sphingolipid Analyses ................................................ 38 2.5. Mitochondrial Lipid Synthesis ....................................... 12 3.8. Lipid Polyester Analysis .............................................. 39 2.6. Triacylglycerol Synthesis ............................................... 15 3.9. Analysis of Cuticular Waxes ....................................... 41 2.7. Lipid Trafficking ............................................................. 18 3.10. Lipidomics ................................................................... 41 2.8. Cuticular Waxes ............................................................ 19 3.11. Strategies for Imaging in Plant Lipid Biology .............. 43 2.9. Biosynthesis of Cutin Polyesters ................................... 22 4. SUMMARY OF ARABIDOPSIS LIPID COMPOSITION .... 46 2.10. Biosynthesis of Suberin Polyesters............................. 24 Tables .................................................................................. 46 2.11. Triacylglycerol Lipases ................................................ 26 Figure .................................................................................. 56 2.12. Fatty Acid β-Oxidation ................................................ 29 REFERENCES ......................................................................... 57 2 of 70 The Arabidopsis Book 1. INTRODUCTION lipids of Arabidopsis, including compositions at the organ, tissue, and subcellular levels. The reactions of Arabidopsis acyl-lipid metabolism require at We do not include in this chapter the very important roles of least 120 enzymatic reactions and more than 600 genes to en- acyl lipids in signaling because this would involve more than 50 code the proteins and regulatory factors involved. These path- additional enzymes and hundreds of genes. We hope other au- ways can be grouped in many ways, but in this chapter we have thors will take up the challenge to include a chapter on Arabidop- organized them into 12 sections based on the types of lipids pro- sis lipid signaling in The Arabidopsis Book. duced and their subcellular localization. To cover such a broad scope of biochemical pathways, structures, and functions is dif- ficult for most researchers, who specialize in one or a few of the A Brief Synopsis and History of Arabidopsis Acyl-Lipid pathways or functions. Therefore, we decided to select a larger Metabolism group of experts who could provide the detailed knowledge and the time needed to identify as many as possible of the Arabidop- Perhaps the best overview of plant acyl-lipid synthesis is provided sis enzymes and genes that are known or suspected to partici- in the textbook chapter by Somerville et al. (2000), which is ex- pate in Arabidopsis acyl-lipid metabolism. The names and con- pected to be updated in 2013. Other more specialized reviews can tact information of each contributor are provided with the sections be found in the Reference section of this chapter, where they are they wrote so that others can contact the appropriate expert with designated with the term “Review” after the reference. corrections, updates, or questions. To better organize all these Based in large part on elegant radiolabeling studies of peas data, we also decided to link this chapter to a web-based commu- and spinach, Roughan and Slack (1982) of New Zealand first pro- nity resource that could provide even more detailed information posed that there are two distinct pathways for membrane synthe- than possible in a chapter of The Arabidopsis Book. This web- sis in higher plants and named these the “prokaryotic pathway” site (ARALIP), http://aralip.plantbiology.msu.edu/, has evolved and the “eukaryotic pathway.” The prokaryotic pathway refers to from the site developed in 2003 and described by Beisson et al. the synthesis of lipids within the plastid. The eukaryotic pathway (2003), which in turn evolved from Mekhedov et al. (2000). Basil refers to the sequence of reactions involved in synthesis of lipids in Shorrosh1 created the new site, the pathway figures, and the un- the endoplasmic reticulum (ER), transfer of some lipids between derlying relational database so that they could be updated easily the ER and the plastid, and further modification of the lipids within to reflect new information. A key feature of the ARALIP website the plastid. Glycerolipids synthesized by the prokaryotic pathway is that each of the figures that describe the pathways includes can be distinguished by the presence of 16:0 at the sn-2 position hyperlinks for all reactions and proteins involved in the pathways. of