An in vivo ¹H and ³¹P NMR investigation of the effect of nitrate on hypoxic metabolism in maize roots

The effect of nitrate on the short-term hypoxic response and recovery of flooded mature maize roots has been investigated in vivo by ¹H and ³¹P NMR and in vitro by ¹H NMR and gas chromatography-mass spectrometry. Employing ¹H NMR in addition to ³¹P NMR extended the number of identifiable compounds in vivo from 4 to 15, while in vitro two-dimensional NMR and gas chromatography-mass spectrometry aided rigorous in vivo ¹H NMR resonance assignments and quantitation of 24 compounds. In the absence of nitrate, the concentrations of key metabolites including alanine, ethanol, γ-aminobutyrate, lactate, succinate, and sucrose changed during 8 h of hypoxia in a manner consistent with reduced tricarboxylic acid cycle activity and diversion to glycolytic fermentation. The pH drop in the cytoplasm during hypoxia was rapid, about 0.2 unit, and diminished quickly upon recovery. Rapid recovery of ethanol, succinate, and sucrose levels was also observed, which indicates a return to normal aerobic metabolism. Although the hypoxic response itself, including pH, was not greatly affected by the presence of nitrate, nitrate reduced the amount of fermentation end products produced, helped maintain a higher free NTP concentration during hypoxia, and increased the rate of overall recovery from hypoxia. These findings suggest the presence of a nitrate-induced maintenance-level respiration in hypoxic maize roots, which helps explain the protection imparted by nitrate to flooded hypoxic maize plants.