MPS 587 - Advanced Plant Biochemistry Course Lipids section guest lecture: Philip Bates Post-doc Browse lab Office: 453 Clark Hall Email: phil_bates@wsu.edu Lecture 9 Lipids I 1. What is a lipid? 2. Fatty acids 3. Lipid classes: structures and functions 4. Lipid analysis
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What is a lipid? Historical definition Lipids are HYDROPHOBIC or AMPHIPATHIC molecules that are typically soluble in organic solvents Lipids are fatty acids (FA) and their derivatives, and substances related biosynthetically or functionally to these compounds. William W. Christie, Scottish Crop Research Institute (http://www.lipidlibrary.co.uk/index.html) Fatty acids are carboxylic acids containing a aliphatic chain of an even (typically) number of carbons. 3
Very little FA accumulate as free fatty acids, most fatty acids are found esterified as oxygen or sulfur (thio) esters or as amide bonds Protein myristoylation Phosphatidylcholine Palmitoyl-CoA 4
Functions of lipids Membranes and compartmentalization Energy storage Protection against pathogens and water loss Caloric content per gram: Fat = 9 Protein = 4 Carbohydrates = 4 (Pollard et al. (2008) Trends in Plant Science 13: 236) Substrates for signaling and defense compounds 5 (Browse J (2005) In Vitamins & Hormones, Vol. 72, Academic Press, p 431)
Fatty acids and derived metabolites Saturated normal-chain fatty acids Systematic name Trivial name Shorthand Designation (http://www.lipidlibrary.co.uk/lipids/fa_sat/index.htm) Nomenclature Number of carbons : number of double bonds ethanoic acetic 2:0 propanoic propionic 3:0 butanoic butyric 4:0 pentanoic 5:0 hexanoic caproic 6:0 heptanoic 7:0 octanoic caprylic 8:0 nonanoic 9:0 decanoic capric 10:0 undecanoic 11:0 dodecanoic lauric 12:0 tridecanoic 13:0 tetradecanoic myristic 14:0 pentadecanoic 15:0 hexadecanoic palmitic 16:0 heptadecanoic margaric 17:0 octadecanoic stearic 18:0 nonadecanoic 19:0 eicosanoic arachidic 20:0 heneicosanoic 21:0 docosanoic behenic 22:0 tricosanoic 23:0 tetracosanoic lignoceric 24:0 pentacosanoic 25:0 hexacosanoic 26:0 heptacosanoic 27:0 octacosanoic 28:0 nonacosanoic 29:0 triacontanoic 30:0 hentriacontanoic 31:0 dotriacontanoic 32:0 6
Monoenoic normal-chain fatty acids (http://www.lipidlibrary.co.uk/lipids/fa_mono/index.htm) Nomenclature of double bonds Δ = count from carboxyl n = ω = count from methyl end Systematic name Trivial name Shorthand designation cis-9-tetradecenoic myristoleic 9-14:1 or 14:1(n-5) Oleic acid, 18:1(n-9) cis-7-hexadecenoic 7-16:1 or 16:1(n-9) cis-9-hexadecenoic palmitoleic 9-16:1 or 16:1(n-7) cis-6-octadecenoic petroselinic 6-18:1 or 18:1(n-12) cis-9-octadecenoic oleic 9-18:1 or 18:1(n-9) cis-11-octadecenoic cis-vaccenic 11-18:1 or 18:1(n-7) cis-11-eicosenoic gondoic 11-20:1 or 20:1(n-9) cis-13-docosenoic erucic 13-22:1 or 22:1(n-9) cis-15-tetracosenoic nervonic 15-24:1 or 24:1(n-9) Eladic acid, 9t-18:1 trans-3-hexadecenoic 3t-16:1 trans-9-octadecenoic elaidic 9t-18:1 trans-11-octadecenoic vaccenic 11t-18:1 Steric acid, 18:0 7
Polyunsaturated fatty acids (http://www.lipidlibrary.co.uk/lipids/fa_conj+/index.htm) n-6 methylene-interrupted n-3 conjugated polymethylene-interrupted Substituted fatty acids hydroxylated (http://www.lipidlibrary.co.uk/lipids/eicplant/index.htm) epoxy furanoid methoxylated (algae) 8
ω-6 and ω-3 Fatty acids are essential in the human diet Essential fatty acids Linoleic acid (18:2 Δ9, 12) α-linolenic acid (18:3 Δ9, 12, 15) Many nutritionally important fatty acids are produced in algae Eicosapentaenoic acid (EPA) 20:5n3 EPA Docosahexaenoic acid (DHA) 22:6n3 Supplemented with algal DHA 9 Wallis JG, Watts JL, Browse J (2002) Polyunsaturated fatty acid synthesis: what will they think of next? Trends in Biochemical Sciences 27: 467-473
Triacylglycerols (TAG) Primary components of fats and oils. Lipid Classes 1. Simple or Neutral lipids Consist of glycerol moiety with each hydroxyl group esterified to a fatty acid. Primary function: energy storage. Diacylglycerols (DAG) http://www.lipidlibrary.co.uk/lipids/whatlip/index.htm Key intermediates in the biosynthesis of triacylglycerols and other lipids. Cellular messengers (generated by hydrolysis of phosphatidylinositol and related metabolites by phospholipase C). Intermediate of the enzymatic hydrolysis of triacylglycerols http://www.lipidlibrary.co.uk/ Lipids/whatlip/index.htm 2-Monoacyl-sn-glycerols (MAG) Formed as intermediates or end-products of the enzymatic hydrolysis of triacylglycerols. 10 http://www.lipidlibrary.co.uk/ Lipids/whatlip/index.htm
mol % mol % Effect of cis double bonds on lipid properties http://www.scientificpsychic.com/fitness/fattyacids1.html 40 35 30 25 20 15 10 5 0 Cocoa butter 16:0 18:0 18:1 18:2(n-6) 18:3(n-3) Physical state of purified plant oils (TAG) Solid 80 70 60 50 40 30 20 10 0 Olive oil 16:0 18:0 18:1 18:2(n-6) 18:3(n-3) Liquid 11
Sterols and sterol esters More information in lectures about isoprenoids. Stigmasterol http://www.lipidlibrary.co.uk/lipids/plant_st/index.htm Tocopherols More information in lectures about isoprenoids. http://www.lipidlibrary.co.uk/lipids/whatlip/index.htm Surface lipids Epicuticular Waxes (not discussed here) Cutins (not discussed here) 12
2. Complex or polar lipids (membrane lipids) Glycerolphospholipids Polar glycerolipid general structure Phosphatidylglycerol (PG) http://www.lipidlibrary.co.uk/lipids/whatlip/index.htm Phosphatidylcholine (PC) Phosphatidylethanolamine (PE) Glycerol backbone sn-1 and sn-1 FA sn-3 polar headgroup Phosphatidylserine (PS) Phosphatidylinositol (PI) 13
Phosphatidic acid (PA) Intermediate in glycerolipid synthesis Signaling molecule generated by lipase digestion of phospholipids Lyso-phosphatidic acid (LPA) Intermediate in glycerolipid synthesis Lyso-lipids Lyso-phosphatidycholine acid (LPC) Intermediate in acyl editing Exclusively in mitochondria. Cardiolipins (CL) http://www.lipidlibrary.co.uk/lipids/dpg/index.htm 14
Non-phosphorous containing glycerolipids Galactolipids Primarily in plant plastids. http://www.lipidlibrary.co.uk/lipids/whatlip/index.htm MGDG DGDG Exclusively in plant plastids. Sulfolipid SQDG http://www.lipidlibrary.co.uk/lipids/whatlip/index.htm 15
Glucosylceramides / Sphingolipids (Sperling & Heinz (2003) Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1632: 1) non-glycerol based membrane lipids Also involved in signaling and stress responses Long chain bases (LCB) Basic ceramide structure and modifications 16
3. Membrane lipid structure/function (Melser et al. (2011) Plant Cell Reports 30: 177) Lipid Rafts As a Membrane-Organizing Principle. (Lingwood & Simons (2010) Science 327: 46) 17
Liquid crystalline phase due to fatty acid unsaturation Membrane dynamics allows compartmentalization and trafficking Fatty acid desaturase mutants are more susceptible to cold Li-Beisson et. al. (2010) In The Arabidopsis Book 8:e0133. doi:10.1199/tab.0133 Figure 8 (Miquel et al. (1993) Proceedings of the National Academy of Sciences of the USA 90: 6208) 18
Lipid abundance Li-Beisson et. al. (2010) In The Arabidopsis Book 8:e0133. doi:10.1199/tab.0133 Figure 21. Li-Beisson et. al. (2010) In The Arabidopsis Book 8:e0133. doi:10.1199/tab.0133 Figure 22. 19
Lipid class composition in different tissues Headgroup Maize (leaves) Wheat (roots) Soybean (seeds) Phospholipids PC 6 50 1-3 PE 3 35 1-3 PG 7 4 1-3 PI 1 6 1-3 CL 1 0 1-3 Galactolipids MGDG 42 1-5 0.4-1.0 DGDG SQDG 31 5 1-5 1-5 0.4-1.0 0.4-1.0 TAG 0.2-1.0 1-5 90-95 20
Lipid class composition in different organelles Headgroup Chloroplast (thylakoid) Mitochondrion (inner membrane) Plasma membrane Phospholipids PC 3 27 32 PE 0 29 46 PG 9 0 0 PI 1 0 19 PS CL 0 0 25 20 0 0 Galactolipids MGDG 51 0 0 DGDG SQDG 26 7 1-2 0 0 0 21
Fatty acid content/composition of Arabidopsis tissues Li-Beisson et. al. (2010) In The Arabidopsis Book 8:e0133. doi:10.1199/tab.0133. Table 4. 22
Lipid analysis General analysis outline Analysis references 1. Lipid extraction Separate lipids from aqueous soluble material with organic solvents 2. Separate/determine lipid classes Thin-Layer Chromatography (TLC) HPLC Mass spectrometry* (LC-MS/MS) Lipidomics 3. Determination of fatty acid content Convert lipid classes to fatty acid methyl esters (FAME) and quantify by GC-FID Mass spectrometry* (LC-MS 3 ) Lipidomics Li-Beisson Y et. al. (2010) Acyl-Lipid Metabolism. The Arabidopsis Book 8:e0133. doi:10.1199/tab.0133 http://www.bioone.org/doi/full/10.1199/ tab.0133 Christie WW (2003) Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids, Ed 3rd. The Oily Press an imprint of PJ Barnes & Associates, Bridgwater, England http://lipidlibrary.aocs.org/ Kansas Lipidomics Research Center http://www.k-state.edu/lipid/lipidomics/ 23
Lipid analysis mol % TLC separation of polar lipids GC-FID of individual lipid classes Neutral lipids MGDG Make FAME PG DGDG SQDG PS PI PE PC 50 45 40 35 30 25 20 15 10 5 0 Developing Arabidopsis seed FA compositions PC PE TAG 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:2 22:1 Arabidopsis: Leaf Seed 24
Lipid analysis http://www.k-state.edu/lipid/lipidomics/profiling.htm Mass spectrometry analysis of lipids Lipid molecular species Number of total FA carbons : number of total double bonds (Welti et al. (2002) Journal of Biological Chemistry 277: 31994) 25