Phosphonate analogs of 2-oxoglutarate perturb metabolism and gene expression in illuminated Arabidopsis leaves

Abstract

Although the role of the 2-oxoglutarate dehydrogenase complex (2-OGDHC) has previously been demonstrated in plant heterotrophic tissues its role in photosynthetically active tissues remains poorly understood. By using a combination of metabolite and transcript profiles we here investigated the function of 2-OGDHC in leaves of Arabidopsis thaliana via use of specific phosphonate inhibitors of the enzyme. Incubation of leaf disks with the inhibitors revealed that they produced the anticipated effects on the in situ enzyme activity. In vitro experiments revealed that succinyl phosphonate (SP) and a carboxy ethyl ester of SP are slow-binding inhibitors of the 2-OGDHC. Our results indicate that the reduced respiration rates are associated with changes in the regulation of metabolic and signaling pathways leading to an imbalance in carbon-nitrogen metabolism and cell homeostasis. The inducible alteration of primary metabolism was associated with altered expression of genes belonging to networks of amino acids, plant respiration, and sugar metabolism. In addition, by using isothermal titration calorimetry we excluded the possibility that the changes in gene expression resulted from an effect on 2-oxoglutarate (2OG) binding to the carbon/ATP sensing protein PII. We also demonstrated that the 2OG degradation by the 2-oxoglutarate dehydrogenase strongly influences the distribution of intermediates of the tricarboxylic acid (TCA) cycle and the GABA shunt. Our results indicate that the TCA cycle activity is clearly working in a non-cyclic manner upon 2-OGDHC inhibition during the light period.

Keywords: 2-oxoglutarate; 2-oxoglutarate dehydrogenase; GABA; PII; TCA cycle; phosphonate inhibitors.

Figure 1

Inhibition of the 2-oxoglutarate dehydrogenase complex activity by SP and CESP. (A) Incubation of leaf disks with phosphonate analogs inhibit the activity of the 2-OGDHC. Arabidopsis thaliana leaf disks were incubated in 10 mM MES-KOH (pH 6.5) with 100 μM SP (white circle) or CESP (black triangle) for up to 4 h in similar light growth conditions. The control (black circles) was incubated in the absence of inhibitor. At the times indicated, the leaf disks were washed with 10 mM MES-KOH (pH 6.5) to remove excess inhibitors and then homogenized. 2-OGDHC activity of the extracts was measured in the standard assay medium without the inhibitors. Each value is the mean ± SE of six biological replicates. (B) Respiration of leaf disks incubated in the absence (black bars) or presence of 100 μM SP (gray bars) or CESP (dark gray bars). Freshly prepared leaf disks were transferred into the temperature controlled measuring chamber of an oxygen electrode containing 1 mL of 10 mM MES-KOH, pH 6.5. Each determination was performed in six biological replicates, and data presented are means ± SE of these 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 2

Effects of the inhibition of the 2-oxoglutarate dehydrogenase complex activity on metabolite levels. Following 4 h incubation of Arabidopsis leaf disks in exactly similar light growth conditions in the absence (white bars) or presence of 50 μM SP (dark bars), 100 μM SP (dark gray bars), 50 μM CESP (gray bars), or 100 μM CESP (light gray bars) levels of protein (A), starch (B), total amino acids (C), nitrate (D), malate (E), and fumarate (F) were measured. Values are means ± SE of six independent samplings. Asterisk indicates values that were determined by the Student’s t-test to be significantly different (P < 0.05) from the control treatment. FW, fresh weight.

Figure 3

Pyridine nucleotide levels and ratios in Arabidopsis leaves following inhibition of 2-OGDHC by phosphonate analogous. Following 4 h incubation of Arabidopsis leaf disks in exactly similar light growth conditions in the absence (white bars) or presence of 50 μM SP (dark bars), 100 μM (dark gray bars), 50 μM CESP (gray bars) or 100 μM CESP (light gray bars) levels of pyridine nucleotide [NADH (A), NAD⁺ (B), NADPH (D), and NADP⁺ (E)] and ratios [NADH/NAD⁺ (C) and NADPH/NADP⁺ (D)] were measured. Values are means ± SE of six independent samplings. Asterisk indicates values that were determined by the Student’s t-test to be significantly different (P < 0.05) from the control treatment. FW, fresh weight.

Figure 4

Heat map representing the changes in relative metabolite contents of treated and control Arabidopsis leaf disks. Leaf disks were cut directly from 4-week-old plants, washed three times with 10 mM MES-KOH (pH 6.5), and then incubated during up to 4 h in 10 mM MES-KOH buffer (pH 6.5) containing 2.0 mM glucose and 100 μM of SP or CESP. Metabolites were determined as described in the Section “Materials and Methods.” Data are normalized with respect to the mean response calculated for the control at 2 h. The full data sets from these metabolic profiling studies are available as Table A1 in Appendix including statistical treatment. Green and red represent a decrease and an increase of metabolite content, respectively, in the SP-treated samples with respect to the control samples. The color scale used is reproduced in the Figure.

Figure 5

Overview of the MapMan visualization of differences in transcript levels between control samples and SP-treated samples. Genes associated with metabolic pathways were analyzed by the MapMan software (http://mapman.gabipd.org/web/guest/mapman). Blue and red represent a decrease and an increase of expression, respectively, in the SP-treated samples with respect to the control samples. The color scale used is reproduced in the Figure. Fully expanded leaves of 4-week-old plants were harvested at the middle of the light period, washed three times with 10 mM MES-KOH (pH 6.5), and then incubated in presence or absence of 100 μM of SP during 4 h in 10 mM MES-KOH buffer (pH 6.5) containing 2.0 mM glucose.

Figure 6

Isothermal titration profiles comparing binding of 2OG to H6PII-tp in the absence and presence of phosphonate analogs or ATP. The upper panel shows the raw data in the form of the heat effect during titration of 120 μm PII with 2OG (5.0–500 μm) and ATP in the presence of SP (A), CESP (B) and in presence of SP but without ATP (C). The lower panel shows the experimental data for the titration of PII plotted in the derivative format, i.e., the binding isotherm, and the best fit curve.

Figure A1

Inhibition of the 2-oxoglutarate dehydrogenase complex activity by SP and PESP.

Figure A2

MapMan visualization of differences in transcript levels between control samples and SP-treated samples.