Inhibition of 2-oxoglutarate dehydrogenase in potato tuber suggests the enzyme is limiting for respiration and confirms its importance in nitrogen assimilation,

Abstract

The 2-oxoglutarate dehydrogenase complex constitutes a mitochondrially localized tricarboxylic acid cycle multienzyme system responsible for the conversion of 2-oxoglutarate to succinyl-coenzyme A concomitant with NAD(+) reduction. Although regulatory mechanisms of plant enzyme complexes have been characterized in vitro, little is known concerning their role in plant metabolism in situ. This issue has recently been addressed at the cellular level in nonplant systems via the use of specific phosphonate inhibitors of the enzyme. Here, we describe the application of these inhibitors for the functional analysis of the potato (Solanum tuberosum) tuber 2-oxoglutarate dehydrogenase complex. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-oxoglutarate dehydrogenase complex, displaying greater inhibitory effects than a diethyl ester of SP, a phosphono ethyl ester of SP, or a triethyl ester of SP. Incubation of potato tuber slices with the inhibitors revealed that they were adequately taken up by the tissue and produced the anticipated effects on the in situ enzyme activity. In order to assess the metabolic consequences of the 2-oxoglutarate dehydrogenase complex inhibition, we evaluated the levels of a broad range of primary metabolites using an established gas chromatography-mass spectrometry method. We additionally analyzed the rate of respiration in both tuber discs and isolated mitochondria. Finally, we evaluated the metabolic fate of radiolabeled acetate, 2-oxoglutarate or glucose, and (13)C-labeled pyruvate and glutamate following incubation of tuber discs in the presence or absence of either SP or the carboxy ethyl ester of SP. The data obtained are discussed in the context of the roles of the 2-oxoglutarate dehydrogenase complex in respiration and carbon-nitrogen interactions.

Figure 1.

Effects of the concentration of SP (black circles), CESP (white circles), DESP (white triangles), PESP (black squares), and TESP (white squares) on the in vitro activity of 2-oxoglutarate dehydrogenase complex in the potato tuber extracts. The enzyme was assayed in the presence of the indicated concentrations of each inhibitor as described in “Materials and Methods.” Each incubation was performed in four biological replicates, and data presented are means ± se of these replicates. FW, Fresh weight.

Figure 2.

In vitro inhibition of 2-oxoglutarate dehydrogenase complex activity by SP (A) or CESP (B). 2-Oxoglutarate dehydrogenase complex activity was measured in the supernatant following centrifugation of the resultant extract, which was incubated with 10 μm (black circles), 25 μm (white circles), 50 μm (black triangles), or 100 μm (white triangles) SP or CESP for up to 240 s. At the times indicated, the enzymatic reaction was started by the addition of 1 mm sodium 2-oxoglutarate. Each value is the mean of four biological replicates.

Figure 3.

Effects of SP (A) or CESP (B) preincubation with the potato tuber discs on the extracted 2-oxoglutarate dehydrogenase complex activity. Potato tuber discs were incubated in 10 mm MES-KOH (pH 6.5) with 100 μm SP or CESP for up to 4 h. The control (black circles) was incubated in the absence of inhibitor. At the times indicated, the tuber discs were washed with 10 mm MES-KOH (pH 6.5) to remove excess inhibitors and then homogenized. 2-Oxoglutarate dehydrogenase complex activity of the extracts was measured in the standard assay medium without the inhibitors. Each value is the mean ± se of four biological replicates. FW, Fresh weight.

Figure 4.

Respiration of tuber discs incubated in the absence (black bars) or presence of 100 μm SP (gray bars) or CESP (dark gray bars). Freshly prepared potato tuber slices were transferred into the temperature-controlled measuring chamber of an oxygen electrode containing 1 mL of 10 mm MES-KOH, pH 6.5. Each value is the mean ± se of four biological replicates. The asterisks demarcate values that were judged to be significantly different from the control (P < 0.05) following the performance of Student's t tests. FW, Fresh weight.

Figure 5.

¹⁴CO₂ evolution following incubation of potato tuber discs in [1-¹⁴C]2-oxoglutarate (A) or [1,2-¹⁴C]acetate (B) in the absence (black bars) or presence of 100 μm SP (gray bars) or CESP (dark gray bars). Each value is the mean ± se of four biological replicates. The asterisks demarcate values that were judged to be significantly different from the control (P < 0.05) following the performance of Student's t tests.

Figure 6.

Effects of SP or CESP on metabolism of [U-¹⁴C]Glc by potato tuber discs. Tuber discs were preincubated in 10 mm MES-KOH (pH 6.5) containing 2 mm Glc in the absence (control) or presence of varying concentrations of SP or CESP for 1 h, and then [U-¹⁴C]Glc (specific activity of 8.11 MBq mmol⁻¹) was added. Each sample was extracted with boiling ethanol, and the amount of radioactivity in each metabolic fraction was determined as described in “Materials and Methods.” Values are expressed as percentages of the total radiolabel metabolized and are means ± se of three biological replicates. The asterisks demarcate values that were judged to be significantly different from the control (P < 0.05) following the performance of Student's t tests.

Figure 7.

Heat map representing the changes in relative metabolite contents of treated and control tuber discs. Tuber discs were cut directly from growing tubers washed three times with 10 mm MES-KOH (pH 6.5) and then incubated for up to 3 h in 10 mm MES-KOH buffer (pH 6.5) containing 2.0 mm Glc and 100 μm SP or CESP. Metabolites were determined as described in “Materials and Methods.” Data are normalized with respect to the mean response calculated for the control at 1 h; values presented are means of four biological replicates. The asterisks demarcate values that were judged to be significantly different from the control (P < 0.05) at the same time point following the performance of Student's t tests.

Figure 8.

Effects of SP or CESP on nitrate content following incubation of potato tuber discs in the absence (black bars) or presence of 100 μm SP (gray bars) or CESP (dark gray bars). Each value is the mean ± se of four biological replicates. The asterisks demarcate values that were judged to be significantly different from the control (P < 0.05) following the performance of Student's t tests. FW, Fresh weight.

Figure 9.

Redistribution of label following incubation of potato tuber discs in 10 mm [¹³C]Glu or [¹³C]pyruvate in the absence (black bars) or presence of 100 μm SP (gray bars) or CESP (dark gray bars). The substrate is indicated in parentheses following the name of the product. Each value is the mean ± se of four biological replicates. The asterisks demarcate values that were judged to be significantly different from the control (P < 0.05) following the performance of Student's t tests. FW, Fresh weight.