Regulation of Primary Metabolism in Response to Low Oxygen Availability as Revealed by Carbon and Nitrogen Isotope Redistribution

Abstract

Based on enzyme activity assays and metabolic responses to waterlogging of the legume Lotus japonicus, it was previously suggested that, during hypoxia, the tricarboxylic acid cycle switches to a noncyclic operation mode. Hypotheses were postulated to explain the alternative metabolic pathways involved, but as yet, a direct analysis of the relative redistribution of label through the corresponding pathways was not made. Here, we describe the use of stable isotope-labeling experiments for studying metabolism under hypoxia using wild-type roots of the crop legume soybean (Glycine max). [(13)C]Pyruvate labeling was performed to compare metabolism through the tricarboxylic acid cycle, fermentation, alanine metabolism, and the γ-aminobutyric acid shunt, while [(13)C]glutamate and [(15)N]ammonium labeling were performed to address the metabolism via glutamate to succinate. Following these labelings, the time course for the redistribution of the (13)C/(15)N label throughout the metabolic network was evaluated with gas chromatography-time of flight-mass spectrometry. Our combined labeling data suggest the inhibition of the tricarboxylic acid cycle enzyme succinate dehydrogenase, also known as complex II of the mitochondrial electron transport chain, providing support for the bifurcation of the cycle and the down-regulation of the rate of respiration measured during hypoxic stress. Moreover, up-regulation of the γ-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.

Figure 1.

Relative abundance of metabolites in soybean root pieces during a 6-h time course of hypoxia treatment determined with GC-TOF-MS. The relative metabolite levels are normalized to an internal standard (ribitol) and the fresh weight of the samples and are depicted on a primary metabolite map. The gray bars represent the ratio of metabolite levels between hypoxia and normoxia conditions at each time interval. The values are means ± se of six biological replicates. Asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P < 0.05). AlaT, Ala aminotransferase; GABA-T, γ-aminobutyric acid trans-aminase; GOGAT, Glu synthase; ICL, isocitrate lyase; ME, malic enzyme; MS, malate synthase; OGDH, 2-oxoglutarate dehydrogenase; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; SSADH, succinic semialdehyde dehydrogenase.

Figure 2.

Total ¹³C accumulation in primary metabolites in soybean roots following [¹³C]pyruvate feeding for a period of 6 h. Black and gray bars represent normoxia and hypoxia conditions, respectively. All data are given in nmol g⁻¹ fresh weight, and asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P < 0.05). Red and blue color represent the transfer of carbon through the pathways of AlaAT and the GABA shunt, respectively. The black dashed lines indicate that pyruvate and 2OG can participate in different reactions; however, the data are the same. The three carbons donated by pyruvate to Ala are highlighted in red. The two carbons donated by acetyl-CoA to citrate are highlighted in blue, at which point they become randomized and no longer can be traced. Abbreviations not defined in Figure 1 are as follows: GAD, Glu decarboxylase; LDH, lactate dehydrogenase.

Figure 3.

Total ¹³C accumulation in primary metabolites in soybean roots following [¹³C]Glu feeding for a period of 6 h. Black and gray bars represent normoxia and hypoxia conditions, respectively. All data are given in nmol g⁻¹ fresh weight, and asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P < 0.05). Red and blue color represent the transfer of carbon through the pathways of AlaAT and the GABA shunt, respectively. The black dashed line indicates that 2OG can participate in different reactions; however, the data are the same. The five carbons donated by Glu to 2OG are highlighted in red. Abbreviations are as defined in Figures 1 and 2.

Figure 4.

Total ¹⁵N accumulation in primary metabolites in soybean roots following ¹⁵NH₄⁺ feeding for a period of 36 h. Black and gray bars represent normoxia and hypoxia conditions, respectively. All data are given in nmol g⁻¹ fresh weight, and asterisks indicate that these values showed significant differences from the control (normoxia) in Student’s t test (P < 0.05). Red color represent the transfer of nitrogen through the pathways of GOGAT and the GABA shunt. The black dashed line indicates that 2OG can participate in different reactions. GS, Gln synthetase. Other abbreviations are as defined in Figures 1 and 2.

Figure 5.

Respiratory oxygen consumption rates of soybean roots during normoxia and hypoxia conditions. Values are means ± se of at least 45 independent measurements (of freshly excised roots 50 min in buffer). FW, Fresh weight.