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 g-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 g-aminobutyric acid shunt and alanine metabolism explained the accumulation of succinate and alanine during hypoxia.

Oxygen Sensing via the Ethylene Response Transcription Factor RAP2.12 Affects Plant Metabolism and Performance under Both Normoxia and Hypoxia

“…Moreover, the levels of GABA were strongly increased, indicating an activation of the GABA shunt. These alterations in respiratory metabolism resemble the metabolic changes that have been found to occur in response to a decrease in oxygen concentrations in previous work (Geigenberger et al, 2000;van Dongen et al, 2009;Narsai et al, 2011;António et al, 2016). As shown in previous studies, a decrease in internal oxygen concentration in roots, tubers, and other plant tissues leads to an adaptive inhibition of respiration as a proactive response to decrease oxygen consumption and prevent the tissue to drive into anoxia (Geigenberger et al, 2000;Zabalza et al, 2009;Geigenberger, 2014).…”

“…This was accompanied by a strong increase in the levels of many amino acids, such as Val, Ala, Ser, Thr, Met, Leu, Ile, Asp, Lys, Cyt, Pro, Orn, Glu, Gln, GABA, Arg, Phe, and Trp (Fig. 5, B and C), a response that has also been found to be a characteristic of hypoxic conditions in the past Narsai et al, 2011;António et al, 2016). Interestingly, there was also an increase in important metabolites deriving from amino acids, Figure 4.…”

“…5C). These changes in TCA cycle intermediates have been found to be a characteristic response to hypoxic conditions in previous studies (Geigenberger et al, 2000;van Dongen et al, 2009;Narsai et al, 2011;António et al, 2016), indicating an inhibition of succinate dehydrogenase (SDH; also known as complex II of the mitochondrial electron transport chain), bifurcation of the TCA cycle, and inhibition of respiration (Rocha et al, 2010;António et al, 2016.). This was accompanied by a strong increase in the levels of many amino acids, such as Val, Ala, Ser, Thr, Met, Leu, Ile, Asp, Lys, Cyt, Pro, Orn, Glu, Gln, GABA, Arg, Phe, and Trp (Fig.…”

“…8). In previous studies it was already concluded from metabolome profiling analysis that the TCA cycle bifurcates in a parallel oxidative and reductive pathway during low oxygen stress (Rocha et al, 2010), while 13 C-labeling experiments were able to show recently that this modification of the TCA modus is mediated by the inhibition of SDH and the activation the GABA shunt (António et al, 2016). In confirmation to this, characteristic low-oxygen-induced changes in the levels of succinate, fumarate, and GABA have been found in various studies with different plant tissues (Narsai et al, 2011), although it remained unclear how the regulation of the TCA cycle reactions is controlled.…”

Mitochondrial Energy Signaling and Its Role in the Low-Oxygen Stress Response of Plants

“…In animals and plants, succinate accumulates under hypoxia (Rocha et al, 2010;Sweetlove et al, 2010;Narsai et al, 2011;Chouchani et al, 2014;António et al, 2016). In animals, succinate can indirectly stabilize the 1a subunit of the transcriptional regulator hypoxia-inducible factor, and succinylation, which relies on succinyl-CoA as substrate, regulates TCA cycle and glycolytic enzymes (Mills and O'Neill, 2014).…”