THE ROLE OF THE TRIOSE-PHOSPHATE SHUTTLE AND GLYCOLYTIC-INTERMEDIATESIN FATTY-ACID AND GLYCEROLIPID BIOSYNTHESIS IN PEA ROOT PLASTIDS

The capacity of the triose-phosphate shuttle and various combinations of glycolytic intermediates to substitute for the ATP requirement for fatty-acid and glycerolipid biosynthesis in pea (Pisum sativum L.) roo t plastids was assessed. In all cases, ATP gave the greatest rates of fatty-acid and glycerolipid biosynthesis. Rates of up to 66 and 27 nmo l.(mg protein)(-1).h(-1) were observed for the incorporation of acetat e and glycerol-3-phosphate into lipids in the presence of ATP. In the absence of exogenously supplied ATP, the triose-phosphate shuttle gave up to 44 and 33% of the ATP-control activity in promoting fatty-acid and glycerolipid biosynthesis from acetate and glycerol-3-phosphate, r espectively. The optimum shuttle components were 2 mM dihydroxyacetone phosphate (DHAP), 2 mM oxaloacetic acid and 4 mM inorganic phosphate ( referred to as the DHAP shuttle). Glyceraldehyde-3-phosphate, as a shu ttle triose, was approximately 82% as effective as DHAP in promoting f atty-acid synthesis while 2-phosphoglycerate, 3-phosphoglycerate, and phosphoenolpyruvate were only 27-37% as effective as DHAP. When glycol ytic intermediates were used as energy sources for fatty-acid synthesi s, in the absence of both exogenously supplied ATP and the triose-phos phate shuttle, phosphoenolpyruvate, 2-phosphoglycerate, fructose-6-pho sphate and glucose-6-phosphate each gave 48%, 17%, 23% and 17%, respec tively, of the ATP-control activity. Other triose phosphates tested we re much less effective in promoting fatty-acid synthesis. When exogeno usly supplied ATP was supplemented with the DHAP shuttle or glycolytic intermediates, the complete shuttle increased fatty-acid biosynthesis by 37% while DHAP alone resulted in 24% stimulation. Glucose-6-phosph ate, fructose-6-phosphate and glycerol-3-phosphate similarly all impro ved the rates of fatty-acid synthesis by 20-30%. In contrast, 3-phosph oglycerate, 2-phosphoglycerate and phosphoenolpyruvate all inhibited f atty-acid synthesis by approximately 10% each. The addition of the DHA P shuttle and glycolytic intermediates with or without exogenously sup plied ATP caused an increase in the proportion of radioactive oleate a nd a decrease in the proportion of radioactive palmitate synthesized. The use of these alternative energy sources resulted in higher amounts of free fatty acids and triacylglycerol, and lower amounts of diacylg lycerol and phosphatidic acid. The data presented here indicate that A TP is superior in promoting in-vitro fatty-acid biosynthesis in pea ro ot plastids; however, both the triose-phosphate shuttle and glycolytic metabolism can produce some of the ATP required for fatty-acid biosyn thesis in these plastids.