Fatty acid synthesis in pea root plastids is inhibited by the action of long-chain acyl- coenzyme as on metabolite transporters

Abstract

The uptake in vitro of glucose (Glc)-6-phosphate (Glc-6-P) into plastids from the roots of 10- to 14-d-old pea (Pisum sativum L. cv Puget) plants was inhibited by oleoyl-coenzyme A (CoA) concentrations in the low micromolar range (1--2 microM). The IC(50) (the concentration of inhibitor that reduces enzyme activity by 50%) for the inhibition of Glc-6-P uptake was approximately 750 nM; inhibition was reversed by recombinant rapeseed (Brassica napus) acyl-CoA binding protein. In the presence of ATP (3 mM) and CoASH (coenzyme A; 0.3 mM), Glc-6-P uptake was inhibited by 60%, due to long-chain acyl-CoA synthesis, presumably from endogenous sources of fatty acids present in the preparations. Addition of oleoyl-CoA (1 microM) decreased carbon flux from Glc-6-P into the synthesis of starch and through the oxidative pentose phosphate (OPP) pathway by up to 73% and 40%, respectively. The incorporation of carbon from Glc-6-P into fatty acids was not detected under any conditions. Oleoyl-CoA inhibited the incorporation of acetate into fatty acids by 67%, a decrease similar to that when ATP was excluded from incubations. The oleoyl-CoA-dependent inhibition of fatty acid synthesis was attributable to a direct inhibition of the adenine nucleotide translocator by oleoyl-CoA, which indirectly reduced fatty acid synthesis by ATP deprivation. The Glc-6-P-dependent stimulation of acetate incorporation into fatty acids was reversed by the addition of oleoyl-CoA.

Figure 1

The rate of Glc-6-P (100 μm) uptake by pea root plastids incubated in the presence of 5 μm oleoyl-CoA and increasing concentrations of rACBP. Silicone oil centrifugation was used to measure uptake of Glc-6-P. Values represent the average of three experiments with ses.

Figure 2

The synthesis of starch over time from Glc-6-P by pea root plastids. The effect of CoASH (0.3 mm), and rACBP (5 μm) was investigated; no additions were made to the control. All incubations contained ATP (3 mm). [¹⁴C]-Labeled starch was determined by repeated methanolic salt washes and starch synthesis expressed as nanomoles Glc-6-P per unit glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Figure 3

The effect of ATP (3 mm), oleoyl-CoA (abbreviation 18:1-CoA, 2 μm), and BSA (5 μm) on the synthesis of fatty acids from acetate (1 mm) by pea root plastids; no additions were made to the control. Incubations were of 60 min. The synthesis of fatty acids is described as nanomoles acetate per unit GAPDH per hour. Fatty acids were purified by thin-layer chromatography prior to scintillation counting.

Figure 4

The effect of Glc-6-P (1 mm) and oleoyl-CoA (2 μm 18:1-CoA) on the synthesis of fatty acids from [1-¹⁴C]acetate by pea root plastids; no additions were made to the control. Incubations were of 60 min. Fatty acid synthesis is expressed as nanomoles acetate per unit glyceraldehyde 3-phosphate dehydrogenase. Values represent the average of three experiments with ses.

Figure 5

The effect of oleoyl-CoA (18:1-CoA) on the uptake of ATP by pea root plastids. The process was determined using the silicone oil centrifugation technique and is described as nanomoles ATP per unit glyceraldehyde 3-phosphate dehydrogenase. Values represent the average of three experiments with ses.