Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos    

Abstract

The reactions leading to triacylglycerol (TAG) synthesis in oilseeds have been well characterized. However, quantitative analyses of acyl group and glycerol backbone fluxes that comprise extraplastidic phospholipid and TAG synthesis, including acyl editing and phosphatidylcholine-diacylglycerol interconversion, are lacking. To investigate these fluxes, we rapidly labeled developing soybean (Glycine max) embryos with [ 14 C]acetate and [14 C]glycerol. Cultured intact embryos that mimic in planta growth were used. The initial kinetics of newly synthesized acyl chain and glycerol backbone incorporation into phosphatidylcholine (PC), 1,2-sn-diacylglycerol (DAG), and TAG were analyzed along with their initial labeled molecular species and positional distributions. Almost 60% of the newly synthesized fatty acids first enter glycerolipids through PC acyl editing, largely at the sn-2 position. This flux, mostly of oleate, was over three times the flux of nascent [14 C]fatty acids incorporated into the sn-1 and sn-2 positions of DAG through glycerol-3-phosphate acylation. Furthermore, the total flux for PC acyl editing, which includes both nascent and preexisting fatty acids, was estimated to be 1.5 to 5 times the flux of fatty acid synthesis. Thus, recycled acyl groups (16:0, 18:1, 18:2, and 18:3) in the acyl-coenzyme A pool provide most of the acyl chains for de novo glycerol-3-phosphate acylation. Our results also show kinetically distinct DAG pools. DAG used for TAG synthesis is mostly derived from PC, whereas de novo synthesized DAG is mostly used for PC synthesis. In addition, two kinetically distinct sn-3 acylations of DAG were observed, providing TAG molecular species enriched in saturated or polyunsaturated fatty acids.

Compartmentation of Triacylglycerol Accumulation in Plants

“…Rapid radiolabeling studies of soybean embryos with [ 14 C]acetate and [ 14 C]glycerol support the existence of separate pools of DAG for TAG and PC biosynthesis (34), a conclusion that was suggested by a number of studies with different plant systems in vitro (24). Labeling embryos with acetate over a short time course (minutes) labels newly synthesized FAs exported from plastids, whereas labeling with glycerol over these same time scales reflects the de novo assembly of glycerolipids by acylation of G3P (34). Comprehensive molecular species characterization and kinetic analyses of these short-term radiolabeling data implicate at least two pools of DAG: one utilized for de novo PC synthesis and one utilized for TAG synthesis (34).…”

“…Alternatively, it is possible that acyl groups are incorporated into phosphatidylcholine (PC) in the plastid envelope and then transported to the ER. Results from rapid radiolabeling studies indicate that PC is the first glycerolipid labeled with newly synthesized FAs (33,34). This may occur via lyso-PC acyltransferase (LPCAT) activity, which has been localized to the chloroplast envelope (35,36).…”

“…In pea leaves (33), where glycerolipid synthesis is primarily for membranes, and in developing soybean (34), where glycerolipid synthesis is overwhelmingly devoted to TAG accumulation, there is a common theme with respect to acyl exchange from PC. In both systems, rapid (within 2 min) [ 14 C]acetate labeling demonstrated that most newly synthesized FAs are first incorporated into PC before any other glycerolipid (33,34). This initial flux of acyl groups into PC bypassed PA and the Kennedy pathway, indicating that the process of rapid acyl exchange occurred independently of net synthesis of phospholipid and TAG in developing embryos.…”

“…Labeling embryos with acetate over a short time course (minutes) labels newly synthesized FAs exported from plastids, whereas labeling with glycerol over these same time scales reflects the de novo assembly of glycerolipids by acylation of G3P (34). Comprehensive molecular species characterization and kinetic analyses of these short-term radiolabeling data implicate at least two pools of DAG: one utilized for de novo PC synthesis and one utilized for TAG synthesis (34). The utilization of PC by plants as the substrate for ER desaturases (65) and as an intermediate in TAG biosynthesis provides a mechanism to increase the presence of polyunsaturated FA in TAG.…”

Role of phosphatidic acid in plant galactolipid synthesis

“…CDP-DAG and DAG are substrates in formation of phospholipids and galactolipids [14]. DAG can also generate TAG ( [83,84]). Conversion to DGPP is an alternative although likely less abundant route [85].…”