In vivo and in vitro metabolomic analysis of anaerobic rice coleoptiles revealed unexpected pathways

The ability of the rice (Oryza sativa L.) seedling to tolerate extended hypoxia during submergence is largely attributed to the biochemical adaptation of its coleoptile. Rice coleoptiles are capable of sustaining ATP production and cytoplasmic pH, unlike flood-sensitive organs, such as maize shoots. Fermentation reactions leading to the production of ethanol, alanine, succinate, and γ-aminobutyrate (GAB) are active in both types of tissues and thus may not account for the difference in tolerance. We have shown previously that rice coleoptiles undergo nitrate reduction and metabolism, which is efficient in alleviating cytoplasmic acidosis and regenerating NAD. Here, we employed ¹³C-2-acetate tracer methods with in vivo ¹³C NMR measurement, including in vivo isotopomer analysis, to probe the tricarboxylic acid (TCA) cycle and interacting pathways in rice coleoptiles during anaerobiosis. We found that the TCA cycle underwent multiple turns based on the metabolic scrambling of ¹³C label patterns in glutamine and malate. The in vivo kinetics of the ¹³C label incorporation into glutamic acid, glutamine, and GAB supports a separate pool of glutamate that was derived from the glutamate dehydrogenase reaction and subsequently decarboxylated to yield GAB. Both reactions consume additional H⁺ and/or NADH. Moreover, the higher rate of ¹³C enrichment at C-3 than C-2 of malate suggests the contribution of the glyoxylate cycle to malate synthesis, which could replenish the TCA cycle carbons diverted to GAB, glutamate, and glutamine synthesis. All of the above reactions contribute to the maintenance of glycolysis for energy production.