BCH 609 Plant Biochemistry Spring 2003 Assignment 1 Page 1 of 5 The main export from the photosynthetic leaves is sucrose, which can be converted and stored as starch, or diverted to triacylglyceride synthesis in oil seeds. Here I shall describe the biochemical changes that would need to be accomplished in order to divert photosynthate from starch synthesis, and result in the synthesis and accumulation tri-tetracosenoin (24:1 containing triacylglyceride or oil) at the expense of 50% starch synthesis. Accumulation of cis-15- tetracosenoic (24:1 n-9), in the seeds of Zea mays could serve as a potential source of biodiesel. The main points to consider are: Diversion of photosynthate (sucrose) away from starch synthesis and towards the precursors of fatty acid synthesis (pyruvate and acetate) Elongation of cis-monounsaturated acyl-CoA substrates by acyl-CoA elongase to generate an increased pool of cis-15-tetracosenoic (24:1 n-9) Incorporation of cis-15-tetracosenoic (24:1 n-9) specifically into triacylglycerides by a diacylglycerol aceyltransferase that has a preference for this substrate When expressing transgenes, tissue specificity needs to be considered, and a promoter that is only turned on in developing embryos is essential Overview In the plastids of higher plants a series of repeated reactions incorporate the acetyl moieties of acetyl-coA into acyl chains 16 or 18 carbons long. This is referred to as the FAS pathway and the enzymes involved are acetyl-CoA carboxylase (ACCase) and the fatty acid synthase complexes (FAS I, II & III). The first step of the pathway is an ATP-dependant carboxylation of acetyl-CoA to form malonyl-CoA. Thioesterases (TE) terminate the fatty acid cycle at 16:0 or 18:0 by hydrolyzing the acyl moiety. Acyl transferase then transfers the fatty acid to glycolipids and releases the ACP carrier protein. The fatty acids are exported to the cytosol as fatty acidCoAs, and further elongation is via elongases located in the endoplasmic reticulum. Page 2 of 5 Diversion of photosynthate from starch synthesis to the precursors of fatty acid synthesis Profiles of starch and lipid accumulation during the embryonic development of oilseed rape shows a net increase in carbon flux to lipid synthesis, and a net decrease to starch. These changes are accompanied by changes in the plastids ability to import metabolites via transporter proteins, and there is an increase in uptake and utilization of pyruvate, accompanied by a decrease of glucose-6-phosphate uptake and utilization for fatty acid synthesis (Eastmond & Rawsthrone, 2002). The developing seeds of the Arabidopsis wri1 mutant exhibit decreased accumulation of oil, and an increased accumulation of carbohydrates, suggesting that the conversion of carbohydrates into the precursors of fatty acid and triacyleglyceride synthesis is in some way adversely affected (Focks & Benning, 1998). Levels of sucrose and glucose are increased in wri1 mutants, relative to wild type, and the precursors of fatty acid biosynthesis (namely acetate and pyruvate) are decreased (Focks & Benning, 1998). This suggests that a block in the conversion of sugars to pyruvate and acetate, and would explain the higher levels of starch synthesis in wri1. Over-expression of WRI1 could potentially result in more hexose sugars being converted to pyruvate and acetate, and ultimately favoring the synthesis of triacylglycerides over starch synthesis. Elongation of cis-monounsaturated acyl-CoA substrates by acyl-CoA elongase Most plant oils contain at least trace amounts of saturated very long chain fatty acids (VLCFAs) of 20-24 carbons in length, and most unsaturated VLCFAs are the result of elongation of oleoylCoA (18:1 n-9) by a membrane bound elongase complex (Voelker & Kinney, 2001). Thus a mechanism to enhance the transcription of these native genes could be an option. Alternatively, transgenic plants could be generated that express genes cloned from plants that are characterized by their accumulation of monounsaturated VLCFAs. β-ketoacyl-CoA synthase is a long chain condensing enzyme that has been purified from Jojoba seeds, which are characterized by oil rich in unsaturated acyl chains 20-24 carbons long. Transgenic Canola seeds, expressing the βketoacyl-CoA synthase gene (KCS1) from jojoba, exhibited accumulation of VLCFAs (Voelker & Kinney, 200). A related gene from Arabidopsis, when expressed in soybean, resulted in the formation of unsaturated fatty acids 20-22 carbons in length at the expense of 16:0, and when Page 3 of 5 FAE1 is co-expressed along with a Δ5 desaturase 20:1 is formed and accounts for approximately 20% of the triacylglyceride (Voelker & Kinney, 2001). Developing seeds of honesty (Lunaria annua) synthesize very-long-chain acyl-CoA thioesters in a manner similar to mammalian systems, via condensation of an acyl-CoA with malonyl-CoA yielding beta-ketoacyl-CoA, which is reduced to beta-hydroxyacyl-CoA, the latter dehydrated to trans-2-enoyl-CoA that is finally reduced to very-long-chain acyl-CoA (Fehling & Mukherjee, 1991). The acyl-CoA elongase does not exhibit any pronounced specificity for any of the saturated (14:0 to 20:0) or (n - 9) cismonounsaturated (14:1 to 22:1) acyl-CoA substrates, although both the saturated and monounsaturated acyl-CoA substrates having chain lengths of C18 and C20 are elongated somewhat faster (Fehling & Mukherjee, 1991). Incorporation of cis-15-tetracosenoic (24:1 n-9) specifically into triacylglycerides Triacylglycerides are synthesized via the Kennedy pathway located in the ER, and TG accumulates in structures known as oil bodies. This pathway is comprised of 3-acyltransferases and a phosphatase. The first 2 acyltransferases transfer a fatty acid from fatty acid-CoA to the sn-1 and the sn-2 positions on the glycerol moiety. Diacylglycerols acyltransferases esterifies fatty acids at the Sn-3 position and are unique to TG biosynthesis, and the only committed step. If diacylglycerol aceyltransferases that show preference for 24:1 could be engineered into maize plants then only 24:1 containing triacylglycerides would be produced, and the composition of membrane lipids would be unaffected. Tissue specificity In contrast to animals, which transport lipids in the form of high- and low-density lipoproteins (HDL & LDL), plants cells each make their own lipids. Thus transgenic plants can be generated that express tissue specific enzymes, and only synthesize the desired products in the developing seeds. Page 4 of 5 References Eastmond P J & Rawsthrone S (2002) “Coodrinate Changes in Carbon Partitioning and Plastidial Metabolism during the Development of Oilseed Rape Embryos” Plant Physiology: 122; 767-774 Fehling E, Mukherjee KD (1991) “Acyl-CoA elongase from a higher plant (Lunaria annua): metabolic intermediates of very-long-chain acyl-CoA products and substrate specificity” Biochim Biophys Acta: 1082; 239-46 Focks N & Benning C1998) ”wrinkled 1: A novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism” Plant Physiology: 118; 91-101 Thelen J J & Ohlrogge (2002) “Metabolic Engineering of Fatty Acid Biosynthesis in Plants” Metabolic Engineering: 4; 12-21 Voelker T & Kinney A J (2001) ”Variations in the Biosynthesis of Seed-Storage Lipids” Annual Review of Plant Physiology and Plant Molecular Biology: 52; 335-361. B.B. Buchanan, W. Gruissem and R.L. Jones, eds.,(2000) Biochemistry & Molecular Biology of Plants ASPP Page 5 of 5